Methods and materials relating to novel polypeptides and polynucleotides

ABSTRACT

The invention provides novel polynucleotides and polypeptides encoded by such polynucleotides and mutants or variants thereof that correspond to the novel polynucleotides and polypeptides. Other aspects of the invention include vectors containing processes for producing novel polypeptides, and antibodies specific for such polypeptides.

Related subject matter is disclosed in the following co-owned,co-pending applications:

-   1) U.S. application Ser. No. 10/005,499, filed Dec. 3, 2001,    entitled “Methods and Materials Relating to Novel Secreted    Adiponectin-like Polypeptides and Polynucleotides”, Attorney Docket    No. HYS-46, which is a continuation-in-part application of PCT    Application Serial No. PCT/US00/35017 filed Dec. 22, 2000 entitled    “Novel Contigs Obtained from Various Libraries”, Attorney Docket No.    784CIP3A/PCT, which in turn is a continuation-in-part application of    U.S. application Ser. No. 09/552,317 filed Apr. 25, 2000 entitled    “Novel Contigs Obtained from Various Libraries”, Attorney Docket No.    784CIP, which in turn is a continuation-in-part application of U.S.    application Ser. No. 09/488'725 filed Jan. 21, 2000 entitled “Novel    Contigs Obtained from Various Libraries”, Attorney Docket No. 784;    PCT application Serial No. PCT/US00/34263, filed Dec. 22, 2000    entitled “Novel Nucleic Acids and Polypeptides”, Attorney Docket No.    784CIP2-2F/PCT, which in turn is a continuation-in-part application    of U.S. application Ser. No. 09/620,312 filed Jul. 19, 2000 entitled    “Novel Nucleic Acids and Polypeptides”, Attorney Docket No.    784CIP2B; PCT Application Serial No. PCT/US01/03800 filed Feb. 5,    2001 entitled “Novel Contigs Obtained from Various Libraries”,    Attorney Docket No. 787CIP3/PCT, which in turn is a    continuation-in-part application of U.S. application Ser. No.    09/560,875 filed Apr. 27, 2000 entitled “Novel Contigs Obtained from    Various Libraries”, Attorney Docket No. 787CIP, which in turn is a    continuation-in-part application of U.S. application Ser. No.    09/496,914 filed Feb. 3, 2000 entitled “Novel Contigs Obtained from    Various Libraries”, Attorney Docket No. 787; PCT application Serial    No. PCT/US01/04098, filed Feb. 5, 2001 entitled “Novel Nucleic Acids    and Polypeptides”, Attorney Docket No. 787CIP2-2G/PCT, which in turn    is a continuation-in-part application of U.S. application Ser. No.    09/598,075 filed Jun. 20, 2000 entitled “Novel Nucleic Acids and    Polypeptides”, Attoreny Docket No. 787CIP2G; PCT Application Serial    No. PCT/US01/08631 filed Mar. 30, 2001 entitled “Novel Contigs    Obtained from Various Libraries”, Attorney Docket No. 790CIP3/PCT,    which in turn is a continuation-in-part application of U.S.    application Ser. No. 09/649,167 filed Aug. 23, 2000 entitled “Novel    Contigs Obtained from Various Libraries”, Attorney Docket No.    790CIP, which in turn is a continuation-in-part application of U.S.    application Ser. No. 09/540,217 filed Mar. 31, 2000 entitled “Novel    Contigs Obtained from Various Libraries”, Attorney Docket No. 790;    U.S. application Ser. No. 09/728,952 filed Nov. 30, 2000 entitled    “Novel Nucleic Acids and Polypeptides”, Attorney Docket No. 799; and    U.S. Provisional application Ser. No. 60/306,971 filed Jul. 21, 2001    entitled “Novel Nucleic Acids and Polypeptides”, Attorney Docket No.    805;-   2) U.S. Application Ser. No. 60/341,362, filed Dec. 17, 2001,    entitled “Methods and Materials Relating to Novel Serpin-like    Polypeptides and Polynucleotides,” Attorney Docket No. HYS-47, which    is related to PCT Application Serial No. PCT/US01/08631, filed Mar.    30, 2001, entitled “Novel Contigs Obtained from Various Libraries,”    Attorney Docket No. 790CIP3/PCT, which in turn is a    continuation-in-part application of U.S. application Ser. No.    09/649,167, filed Aug. 23, 2000, entitled “Novel Contigs Obtained    from Various Libraries,” Attorney Docket No. 790CIP, which in turn    is a continuation-in-part application of U.S. application Ser. No.    09/540,217 filed Mar. 31, 2000 entitled “Novel Contigs Obtained from    Various Libraries,” Attorney Docket No. 790;-   3) U.S. Application Ser. No. 60/379,875 filed May 10, 2002 entitled    “Novel Nogo-Receptor-like Protein Materials and Methods,” Attorney    Docket No. HYS-52;-   4) U.S. Application Ser. No. 60/379,834 filed May 10, 2002 entitled    “Methods and Materials Relating to Scavenger Receptor-like    Polypeptides and Polynucleotides,” Attorney Docket No. HYS-54, which    is related to U.S. application Ser. No. 09/687,535 filed Oct. 13,    2000 entitled “Methods and Materials Relating to Scavenger    Receptor-like Polypeptides and Polynucleotides,” Attorney Docket No.    HYS-32;-   5) U.S. Application Ser. No. 60/384,450 filed May 31, 2002 entitled    “Methods and Materials Relating to Neural Immunoglobulin Cell    Adhesion Molecule-like Polypeptides and Polynucleotides,” Attorney    Docket No. HYS-55;-   6) U.S. Application Ser. No. 60/384,665 filed May 31, 2002 entitled    “Methods and Materials Relating to Growth Hormone-like Polypeptides    and Polynucleotides,” Attorney Docket No. HYS-57;-   7) U.S. Application Ser. No. 60/389,715 filed Jun. 17, 2002 entitled    “Methods and Materials Relating to Neutrophil Gelatinase-associated    Lipocalin-like Polypeptides and Polynucleotides,” Attorney Docket    No. HYS-58, which is related to U.S. Application Ser. No. 60/365,384    filed on Mar. 14, 2002 entitled “Novel Nucleic Acids and Secreted    Polypeptides,” Attorney Docket No. 814, which is a    continuation-in-part application of PCT Application Serial No.    PCT/US00/35017 filed Dec. 22, 2000 entitled “Novel Contigs Obtained    from Various Libraries,” Attorney Docket No. 784CIP3/PCT, which in    turn is a continuation-in-part application of U.S. application Ser.    No. 09/552,317 filed Apr. 25, 2000 entitled “Novel Contigs Obtained    from Various Libraries,” Attorney Docket No. 784CIP, which in turn    is a continuation-in-part application of U.S. application Ser. No.    09/488,725 filed Jan. 21, 2000 entitled “Novel Contigs Obtained from    Various Libraries,” Attorney Docket No. 784; and is a    continuation-in-part application of PCT Application Serial No.    PCT/US00/34263 filed Dec. 26, 2000 entitled “Novel Nucleic Acids and    Polypeptides,” Attorney Docket No. 784CIP2-2F/PCT, which is a    continuation-in-part application of U.S. application Ser. No.    09/620,312 filed Jul. 19, 2000, entitled “Novel Nucleic Acids and    Polypeptides,” Attorney Docket No. 784CIP2B, which in turn is a    continuation-in-part application of U.S. application Ser. No.    09/552,317 filed Apr. 25, 2000, entitled “Novel Contigs Obtained    from Various Libraries,” Attorney Docket No. 784CIP, which in turn    is a continuation-in-part application of U.S. application Ser. No.    09/488,725 filed Jan. 21, 2000, entitled “Novel Contigs Obtained    from Various Libraries,” Attorney Docket No. 784;-   8) U.S. Application Ser. No. 60/393,722 filed Jul. 2, 2002 entitled    “Methods and Materials Relating to Novel Mucolipin-like Polypeptides    and Polynucleotides,” Attorney Docket No. HYS-60;-   9) U.S. Application Ser. No. 60/390,531 filed Jun. 21, 2002 entitled    “Methods and Materials Relating to Peroxidasin-like Polypepides and    Polynucleotides,” Attorney Docket No. HYS-61;-   10) U.S. Application Ser. No. 60/391,326 filed Jun. 24, 2002    entitled “Methods and Materials Relating to Synaptic Associated    Protein 90/Postsynaptic Density Protein 95 kDa-associated    Protein-like Polypeptides and Polynucleotides,” Attorney Docket No.    HYS-62; all of which are herein incorporated by reference in their    entirety.

1. BACKGROUND

1.1 Technical Field

The present invention provides novel polynucleotides and proteinsencoded by such polynucleotides, along with uses for thesepolynucleotides and proteins, for example in therapeutic, diagnostic andresearch methods.

1.2 Background Art

Technology aimed at the discovery of protein factors (including e.g.,cytokines, such as lymphokines, interferons, CSFs, chemokines, andinterleukins) has matured rapidly over the past decade. The now routinehybridization cloning and expression cloning techniques clone novelpolynucleotides “directly” in the sense that they rely on informationdirectly related to the discovered protein (i.e., partial DNA/amino acidsequence of the protein in the case of hybridization cloning; activityof the protein in the case of expression cloning). More recent“indirect” cloning techniques such as signal sequence cloning, whichisolates DNA sequences based on the presence of a now well-recognizedsecretory leader sequence motif, as well as various PCR-based or lowstringency hybridization-based cloning techniques, have advanced thestate of the art by making available large numbers of DNA/amino acidsequences for proteins that are known to have biological activity, forexample, by virtue of their secreted nature in the case of leadersequence cloning, by virtue of their cell or tissue source in the caseof PCR-based techniques, or by virtue of structural similarity to othergenes of known biological activity.

Identified polynucleotide and polypeptide sequences have numerousapplications in, for example, diagnostics, forensics, gene mapping,identification of mutations responsible for genetic disorders or othertraits, to assess biodiversity, and to produce many other types of dataand products dependent on DNA and amino acid sequences. Proteins areknown to have biological activity, for example, by virtue of theirsecreted nature in the case of leader sequence cloning, by virtue oftheir cell or tissue source in the case of PCR-based techniques, or byvirtue of structural similarity to other genes of known biologicalactivity. It is to these polypeptides and the polynucleotides encodingthem that the present invention is directed.

2. SUMMARY OF THE INVENTION

This invention is based on the discovery of novel polypeptides, novelisolated polynucleotides encoding such polypeptides, includingrecombinant DNA molecules, cloned genes or degenerate variants thereof,especially naturally occurring variants such as allelic variants,antisense polynucleotide molecules, and antibodies that specificallyrecognize one or more epitopes present on such polypeptides, as well ashybridomas producing such antibodies. The compositions of the presentinvention additionally include vectors such as expression vectorscontaining the polynucleotides of the invention, cells geneticallyengineered to contain such polynucleotides, and cells geneticallyengineered to express such polynucleotides.

The compositions of the invention provide isolated polynucleotides thatinclude, but are not limited to, a polynucleotide comprising thenucleotide sequence set forth in SEQ ID NO 1-4, 6, 14, 16, 25-27, 29,157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303,322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419,421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529,547, 549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606,608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631; or a fragmentthereof that retains a desired biological activity, a polynucleotidecomprising the full length protein coding sequence of SEQ ID NO: 1-4, 6,14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271,273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379,405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506,514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578,580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625,627, 629, or 631 (for example, the open reading frame of SEQ ID NO: 5,15, 28, 160, 186, 215, 241, 272, 302, 323, 348, 355, 378, 408, 420, 444,487, 505, 516, 528, 542, 548, 557, 572, 579, 588, 602, 607, 612, 618,622, 626, or 630); and a polynucleotide comprising the nucleotidesequence of the mature protein coding sequence of any of SEQ ID NO:1-4,6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240,242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377,379, 405-407, 409, 418-419, 421, 441-443, 485486, 488, 503, 504, 506,514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578,580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625,627, 629, or 631. The polynucleotides of the present invention alsoinclude, but are not limited to, a polynucleotide that hybridizes understringent hybridization conditions to (a) the complement of any of thenucleotide sequences set forth in SEQ ID NO: 1-4, 6, 14, 16, 25-27, 29,157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303,322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419,421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529,547, 549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606,608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631; (b) anucleotide sequence encoding any of the amino acid sequences set forthin SEQ D NO: 5, 7-13, 15, 17-24, 28, 30-156, 160, 162-182, 186, 188-213,215, 217-239, 241, 243-270, 272, 274-299, 302, 304-321, 323, 325-344,348, 350-352, 355, 357-376, 378, 380-401, 408, 410-414, 415, 420,422-439, 444-480, 482-484,487,489-501, 505, 507-512, 516, 518-524, 528,530-539, 542, 544-546, 548, 550-553, 557, 559-567, 572, 574, 576, 579,581-584, 588, 590, 596, 602, 604-605, 607, 609-610, 612, 614-615, 618,620, 622, 624, 626, 628, 630, 632, or 634-653; a polynucleotide which isan allelic variant of any polynucleotides recited above having at least70% polynucleotide sequence identity to the polynucleotides; apolynucleotide which encodes a species homolog (e.g. orthologs) of anyof the peptides recited above; or a polynucleotide that encodes apolypeptide comprising a specific domain or truncation of thepolypeptide of SEQ ID NO: 5, 7-13, 15, 17-24, 28, 30-156, 160, 162-182,186, 188-213, 215, 217-239, 241, 243-270, 272, 274-299, 302, 304-321,323, 325-344, 348, 350-352, 355, 357-376, 378, 380-401, 408, 410-414,415, 420, 422-439, 444-480, 482-484, 487, 489-501, 505, 507-512, 516,518-524, 528, 530-539, 542, 544-546, 548, 550-553, 557, 559-567, 572,574, 576, 579, 581-584, 588, 590, 596, 602, 604-605, 607, 609-610, 612,614-615, 618, 620, 622, 624, 626, 628, 630, 632, or 634-653.

A collection as used in this application can be a collection of only onepolynucleotide. The collection of sequence information or uniqueidentifying information of each sequence can be provided on a nucleicacid array. In one embodiment, segments of sequence information areprovided on a nucleic acid array to detect the polynucleotide thatcontains the segment. The array can be designed to detect full-match ormismatch to the polynucleotide that contains the segment. The collectioncan also be provided in a computer-readable format.

This invention further provides cloning or expression vectors comprisingat least a fragment of the polynucleotides set forth above and hostcells or organisms transformed with these expression vectors. Usefulvectors include plasmids, cosmids, lambda phage derivatives, phagemids,and the like, that are well known in the art. Accordingly, the inventionalso provides a vector including a polynucleotide of the invention and ahost cell containing the polynucleotide. In general, the vector containsan origin of replication functional in at least one organism, convenientrestriction endonuclease sites, and a selectable marker for the hostcell. Vectors according to the invention include expression vectors,replication vectors, probe generation vectors, and sequencing vectors. Ahost cell according to the invention can be a prokaryotic or eukaryoticcell and can be a unicellular organism or part of a multicellularorganism.

The compositions of the present invention include polypeptidescomprising, but not limited to, an isolated polypeptide selected fromthe group comprising the amino acid sequence of SEQ ID NO: 5, 7-13, 15,17-24, 28, 30-156, 160, 162-182, 186, 188-213, 215, 217-239, 241,243-270, 272, 274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355,357-376, 378, 380-401, 408, 410-414, 415, 420, 422-439, 444-480,482-484, 487, 489-501, 505, 507-512, 516, 518-524, 528, 530-539, 542,544-546, 548, 550-553, 557, 559-567, 572, 574, 576, 579, 581-584, 588,590, 596, 602, 604-605, 607, 609-610, 612, 614-615, 618, 620, 622, 624,626, 628, 630, 632, or 634-653; or the corresponding full length ormature protein. Polypeptides of the invention also include polypeptideswith biological activity that are encoded by (a) any of thepolynucleotides having a nucleotide sequence set forth in SEQ ID NO:1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240,242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377,379, 405-407, 409, 418419, 421, 441443, 485-486, 488, 503, 504, 506,514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578,580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625,627, 629, or 631; or (b) polynucleotides that hybridize to thecomplement of the polynucleotides of (a) under stringent hybridizationconditions. Biologically or immunologically active variants of any ofthe protein sequences listed as SEQ ID NO: 5, 7-13, 15, 17-24, 28,30-156, 160, 162-182, 186, 188-213, 215, 217-239, 241, 243-270, 272,274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355, 357-376, 378,380-401, 408, 410-414, 415, 420, 422-439, 444480, 482-484, 487, 489-501,505, 507-512, 516, 518-524, 528, 530-539, 542, 544-546, 548, 550-553,557, 559-567, 572, 574, 576, 579, 581-584, 588, 590, 596, 602,604-605,607,609-610, 612, 614-615, 618, 620, 622, 624, 626, 628, 630,632, or 634-653 and substantial equivalents thereof that retainbiological or immunological activity are also contemplated. Thepolypeptides of the invention may be wholly or partially chemicallysynthesized but are preferably produced by recombinant means using thegenetically engineered cells (e.g. host cells) of the invention.

The invention also provides compositions comprising a polypeptide of theinvention. Pharmaceutical compositions of the invention may comprise apolypeptide of the invention and an acceptable carrier, such as ahydrophilic, e.g., pharmaceutically acceptable, carrier.

The invention also relates to methods for producing a polypeptide of theinvention comprising culturing host cells comprising an expressionvector containing at least a fragment of a polynucleotide encoding thepolypeptide of the invention in a suitable culture medium underconditions permitting expression of the desired polypeptide, andpurifying the protein or peptide from the culture or from the hostcells. Preferred embodiments include those in which the protein producedby such a process is a mature form of the protein.

Polynucleotides according to the invention have numerous applications ina variety of techniques known to those skilled in the art of molecularbiology. These techniques include use as hybridization probes, use asoligomers, or primers, for PCR, use in an array, use incomputer-readable media, use for chromosome and gene mapping, use in therecombinant production of protein, and use in generation of antisenseDNA or RNA, their chemical analogs and the like. For example, when theexpression of an mRNA is largely restricted to a particular cell ortissue type, polynucleotides of the invention can be used ashybridization probes to detect the presence of the particular cell ortissue mRNA in a sample using, e.g., in situ hybridization.

In other exemplary embodiments, the polynucleotides are used indiagnostics as expressed sequence tags for identifying expressed genesor, as well known in the art and exemplified by Vollrath et al., Science258:52-59 (1992), as expressed sequence tags for physical mapping of thehuman genome.

The polypeptides according to the invention can be used in a variety ofconventional procedures and methods that are currently applied to otherproteins. For example, a polypeptide of the invention can be used togenerate an antibody that specifically binds the polypeptide. Suchantibodies, particularly monoclonal antibodies, are useful for detectingor quantitating the polypeptide in tissue. The polypeptides of theinvention can also be used as molecular weight markers, and as a foodsupplement.

Methods are also provided for preventing, treating, or ameliorating amedical condition which comprises the step of administering to amammalian subject a therapeutically effective amount of a compositioncomprising a peptide of the present invention and a pharmaceuticallyacceptable carrier.

The methods of the invention also provide methods for the treatment ofdisorders as recited herein which comprise the administration of atherapeutically effective amount of a composition comprising apolynucleotide or polypeptide of the invention and a pharmaceuticallyacceptable carrier to a mammalian subject exhibiting symptoms ortendencies related to disorders as recited herein. In addition, theinvention encompasses methods for treating diseases or disorders asrecited herein comprising the step of administering a compositioncomprising compounds and other substances that modulate the overallactivity of the target gene products and a pharmaceutically acceptablecarrier. Compounds and other substances can effect such modulationeither on the level of target gene/protein expression or target proteinactivity. Specifically, methods are provided for preventing, treating orameliorating a medical condition, including viral diseases, whichcomprises administering to a mammalian subject, including but notlimited to humans, a therapeutically effective amount of a compositioncomprising a polypeptide of the invention or a therapeutically effectiveamount of a composition comprising a binding partner of (e.g., antibodyspecifically reactive for) the polypeptides of the invention. Themechanics of the particular condition or pathology will dictate whetherthe polypeptides of the invention or binding partners (or inhibitors) ofthese would be beneficial to the individual in need of treatment.

According to this method, polypeptides of the invention can beadministered to produce an in vitro or in vivo inhibition of cellularfunction. A polypeptide of the invention can be administered in vivoalone or as an adjunct to other therapies. Conversely, protein or otheractive ingredients of the present invention may be included informulations of a particular agent to minimize side effects of such anagent.

The invention further provides methods for manufacturing medicamentsuseful in the above-described methods.

The present invention further relates to methods for detecting thepresence of the polynucleotides or polypeptides of the invention in asample (e.g., tissue or sample). Such methods can, for example, beutilized as part of prognostic and diagnostic evaluation of disorders asrecited herein and for the identification of subjects exhibiting apredisposition to such conditions.

The invention provides a method for detecting a polypeptide of theinvention in a sample comprising contacting the sample with a compoundthat binds to and forms a complex with the polypeptide under conditionsand for a period sufficient to form the complex and detecting formationof the complex, so that if a complex is formed, the polypeptide isdetected.

The invention also provides kits comprising polynucleotide probes and/ormonoclonal antibodies, and optionally quantitative standards, forcarrying out methods of the invention. Furthermore, the inventionprovides methods for evaluating the efficacy of drugs, and monitoringthe progress of patients, involved in clinical trials for the treatmentof disorders as recited above.

The invention also provides methods for the identification of compoundsthat modulate (i.e., increase or decrease) the expression or activity ofthe polynucleotides and/or polypeptides of the invention. Such methodscan be utilized, for example, for the identification of compounds thatcan ameliorate symptoms of disorders as recited herein. Such methods caninclude, but are not limited to, assays for identifying compounds andother substances that interact with (e.g., bind to) the polypeptides ofthe invention.

The invention provides a method for identifying a compound that binds tothe polypeptide of the present invention comprising contacting thecompound with the polypeptide under conditions and for a time sufficientto form a polypeptide/compound complex and detecting the complex, sothat if the polypeptide/compound complex is detected, a compound thatbinds to the polypeptide of the invention is identified.

Also provided is a method for identifying a compound that binds to apolypeptide of the invention comprising contacting the compound with apolypeptide of the invention in a cell for a time sufficient to form apolypeptide/compound complex wherein the complex drives expression of areporter gene sequence in the cell and detecting the complex bydetecting reporter gene sequence expression so that if thepolypeptide/compound complex is detected a compound that binds to thepolypeptide of the invention is identified.

3. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 5 and adiponectin SEQ ID NO: 402(Hotta et al., Diabetes 50:1126-1133 (2001)).

FIG. 2 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 5 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1).

FIG. 3 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 15 and adiponectin SEQ ID NO:402 (Hotta et al., Diabetes 50:1126-1133 (2001)).

FIG. 4 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 15 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1).

FIG. 5 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 28 and adiponectin SEQ ID NO:402 (Hotta et al., Diabetes 50:1126-1133 (2001)).

FIG. 6 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 28 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1).

FIG. 7 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 160 and adiponectin SEQ ID NO:402 (Hotta et al., Diabetes 50:1126-1133 (2001)).

FIG. 8 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 160 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1).

FIG. 9 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 186 and adiponectin SEQ ID NO:402 (Hotta et al., Diabetes 50:1126-1133 (2001)).

FIG. 10 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 186 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1).

FIG. 11 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 215 and adiponectin SEQ ID NO:402 (Hotta et al., Diabetes 50:1126-1133 (2001)).

FIG. 12 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 215 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1).

FIG. 13 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 241 and adiponectin SEQ ID NO:402 (Hotta et al., Diabetes 50:1126-1133 (2001)).

FIG. 14 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 241 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1).

FIG. 15 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 272 and adipose tissue-specificprotein AdipoQ SEQ ID NO: 403 (Sato et al., J. Biol. Chem.276:28849-28856 (2001)).

FIG. 16 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 272 and adipose tissue-specificprotein AdipoQ SEQ ID NO: 403 (Sato et al., J. Biol. Chem.276:28849-28856 (2001)).

FIG. 17 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 272 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1).

FIG. 18 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 302 and adiponectin SEQ ID NO:402 (Hotta et al., Diabetes 50:1126-1133 (2001)).

FIG. 19 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 302 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1).

FIG. 20 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 323 and adiponectin SEQ ID NO:402 (Hotta et al., Diabetes 50:1126-1133 (2001)).

FIG. 21 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 323 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1).

FIG. 22 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 348 and adiponectin SEQ ID NO:402 (Hotta et al., Diabetes 50:1126-1133 (2001)).

FIG. 23 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 348 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1).

FIG. 24 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 355 and adiponectin SEQ ID NO:402 (Hotta et al., Diabetes 50:1126-1133 (2001)).

FIG. 25 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 355 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1).

FIG. 26 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 378 and adiponectin SEQ ID NO:402 (Hotta et al., Diabetes 50:1126-1133 (2001)).

FIG. 27 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 378 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1).

FIG. 28 shows the BLASTP amino acid sequence alignment of the first highscoring pair (HSP) between Serpin-like polypeptide SEQ ID NO: 408 andSERPINB12 SEQ ID NO: 416 (Askew et al., J. Biol. Chem. 276:49320-49330(2001), herein incorporated by reference in its entirety).

FIG. 29 shows the BLASTP amino acid sequence alignment of the secondhigh scoring pair (HSP) between Serpin-like polypeptide SEQ ID NO: 408and SERPINB12 SEQ ID NO: 416 (Askew et al., J. Biol. Chem.276:49320-49330 (2001), herein incorporated by reference in itsentirety).

FIG. 30 shows the BLASTP amino acid sequence alignment of the first highscoring pair (HSP) between Serpin-like polypeptide SEQ ID NO: 408 andhuman SCCA2 protein SEQ ID NO: 417 (Patent No. DE19742725-A1, hereinincorporated by reference in its entirety).

FIG. 31 shows the BLASTP amino acid sequence alignment of the secondhigh scoring pair (HSP) between Serpin-like polypeptide SEQ ID NO: 408and human SCCA2 protein SEQ ID NO: 417 (Patent No. DE19742725-A1, hereinincorporated by reference in its entirety).

FIG. 32 shows a schematic diagram illustrating the major structuralfeatures of the Nogo receptor, NgR, and the Nogo receptor homolog,NgRHy.

FIG. 33 shows the BLASTP amino acid sequence alignment between theprotein encoded by SEQ ID NO: 419 (i.e. SEQ ID NO: 420), NgRHy, and thehuman NgR (SEQ ID NO: 440).

FIG. 34 shows the BLASTX amino acid sequence alignment between theprotein encoded by SEQ ID NO: 443 (i.e. SEQ ID NO: 444), scavengerreceptor-like polypeptide and mouse macrophage scavenger receptor type I(SEQ ID NO: 481).

FIG. 35 (A, B) shows a BLASTP amino acid sequence alignment betweenneural IgCAM-like polypeptide (SEQ ID NO: 487) and another member of thefamily, mouse PANG (SEQ ID NO: 502).

FIG. 36 shows a BLASTP amino acid sequence alignment between neuralIgCAM-like polypeptide (SEQ ID NO: 505) and bovine NCAM-140 (SEQ ID NO:513).

FIG. 37 shows a multiple amino acid sequence alignment between neuralIgCAM-like polypeptide (SEQ ID NO: 505), neural IgCAM-like polypeptide(SEQ ID NO: 542) and bovine NCAM-140 (SEQ ID NO: 513).

FIG. 38 shows a BLASTP amino acid sequence alignment between neuralIgCAM-like polypeptide (SEQ ID NO: 516) and another member of thefamily, mouse DDM36 (SEQ ID NO: 52).

FIG. 39 (A, B) shows a BLASTP amino acid sequence alignment betweenneural IgCAM-like polypeptide (SEQ ID NO: 530) and another member of thefamily, rat BIG-2 (SEQ ID NO: 540).

FIG. 40 shows a BLASTP amino acid sequence alignment between growthhormone-like polypeptide (SEQ ID NO: 548) and human chorionicsomatomammotropin hormone-like 1, isoform 3 precursor (SEQ ID NO: 554).

FIG. 41 shows a BLASTP amino acid sequence alignment between growthhormone-like polypeptide (SEQ ID NO: 548) and human chorionicsomatomammotropin hormone-like 1, isoform 5 precursor (SEQ ID NO: 555).

FIG. 42 shows a BLASTP amino acid sequence alignment between growthhormone-like polypeptide (SEQ ID NO: 557) and human chorionicsomatomammotropin hormone 1, isoform 2 precursor (SEQ ID NO: 568).

FIG. 43 shows a BLASTP amino acid sequence alignment between growthhormone-like polypeptide (SEQ ID NO: 557) and human growth hormone 2,isoform 2 precursor (SEQ ID NO: 569).

FIG. 44 shows a multiple sequence alignment between NGAL-likepolypeptides (SEQ ID NO: 572 and 579) and other members of the family:(SEQ ID NO: 585 and 586, respectively).

FIG. 45 shows a BLASTP amino acid sequence alignment of mucolipin-likepolypeptide (SEQ ID NO: 588) and human mucolipin 1 (SEQ ID NO: 592).

FIG. 46 (A, B) shows a multiple amino acid sequence alignment ofmucolipin-like polypeptide (SEQ ID NO: 588) and other members of thefamily: mouse mucolipin 2 (SEQ ID NO: 591), human mucolipin 1 (SEQ IDNO: 592), human mucolipin 3 (SEQ ID NO: 593), C. elegans CUP-5 (SEQ IDNO: 595).

FIG. 47 shows an alignment of the conserved serine lipase active sitebetween mucolipin-like polypeptide (SEQ ID NO: 596) and mucolipin 1 (SEQID NO: 597), as well as other lipolytic enzymes: H. liph triacylglycerollipase, hepatic precursor (SEQ ID NO: 598), H. liph lipoprotein lipaseprecursor (SEQ ID NO: 599), and H. lcat phosphatidylcholine-sterolacyltransferase precursor (SEQ ID NO: 600).

FIG. 48 (A, B) shows a BLASTP amino acid sequence alignment between aperoxidasin-like polypeptide (SEQ ID NO: 602) and another member of thefamily, human peroxidasin-like protein MG50 (SEQ ID NO: 616).

FIG. 49 (A, B, C) shows a multiple sequence alignment betweenperoxidasin-like polypeptides SEQ ID NO: 602, 618, 622, and 626.

FIG. 50 (A, B) shows a BLASTP amino acid sequence alignment between asecond peroxidasin-like polypeptide (SEQ ID NO: 607) and another memberof the family, human peroxidasin-like protein MG50 (SEQ ID NO: 616).

FIG. 51 (A, B) shows a BLASTP amino acid sequence alignment between athird peroxidasin-like polypeptide (SEQ ID NO: 612) and another memberof the family, human peroxidasin-like protein MG50 (SEQ ID NO: 616).

FIG. 52 (A, B) shows a BLASTP amino acid sequence alignment betweenSAPAP-like polypeptide (SEQ ID NO: 630) and rat SAPAP3 (SEQ ID NO: 633).

4. DETAILED DESCRIPTION OF THE INVENTION

Table 1 is a correlation table of the novel polynucleotide sequences(14, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240,242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377,379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506,514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578,580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625,627, 629, and 631) and the novel polypeptides (5, 7-13, 15, 17-24, 28,30-156, 160, 162-182, 186, 188-213, 215, 217-239, 241, 243-270, 272,274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355, 357-376, 378,380-401, 408, 410-414, 415, 420, 422-439, 444-480, 482-484, 487,489-501, 505, 507-512, 516, 518-524, 528, 530-539, 542, 544-546, 548,550-553, 557, 559-567, 572, 574, 576, 579, 581-584, 588, 590, 596, 602,604-605, 607, 609-610, 612, 614-615, 618, 620, 622, 624, 626, 628, 630,632, and 634-653) and the corresponding SEQ ID NO: in which the sequencewas filed in the following priority U.S. patent Applications bearing theserial numbers of: Ser. No. 10/005,499 filed on Dec. 3, 2001, 60/341,362filed on Dec. 17, 2001, 60/379,875 filed on May 10, 2002, 60/379,834filed May 10, 2002, 60/384,450 filed on May 31, 2002, 60/384,665 filedon May 31, 2002, 60/389,715 filed on Jun. 17, 2002, 60/393,722, filed onJul. 2, 2002, 60/390,531 filed on Jun. 21, 2002, and 60/391,326 filed onJun. 24, 2002. TABLE 1 Identification of Priority Application thatsequence was filed (Attorney SEQ ID NO: Docket No._SEQ ID NO.) * 1HYS-46_1 2 HYS-46_2 3 HYS-46_3 4 HYS-46_4 5 HYS-46_5 6 HYS-46_6 7HYS-46_7 8 HYS-46_8 9 HYS-46_9 10 HYS-46_10 11 HYS-46_11 12 HYS-46_12 13HYS-46_13 14 HYS-46_14 15 HYS-46_15 16 HYS-46_16 17 HYS-46_17 18HYS-46_18 19 HYS-46_19 20 HYS-46_20 21 HYS-46_21 22 HYS-46_22 23HYS-46_23 24 HYS-46_24 25 HYS-46_25 26 HYS-46_26 27 HYS-46_27 28HYS-46_28 29 HYS-46_29 30 HYS-46_30 31 HYS-46_31 32 HYS-46_32 33HYS-46_33 34 HYS-46_34 35 HYS-46_35 36 HYS-46_36 37 HYS-46_37 38HYS-46_38 39 HYS-46_39 40 HYS-46_40 41 HYS-46_41 42 HYS-46_42 43HYS-46_43 44 HYS-46_44 45 HYS-46_45 46 HYS-46_46 47 HYS-46_47 48HYS-46_48 49 HYS-46_49 50 HYS-46_50 51 HYS-46_51 52 HYS-46_52 53HYS-46_53 54 HYS-46_54 55 HYS-46_55 56 HYS-46_56 57 HYS-46_57 58HYS-46_58 59 HYS-46_59 60 HYS-46_60 61 HYS-46_61 62 HYS-46_62 63HYS-46_63 64 HYS-46_64 65 HYS-46_65 66 HYS-46_66 67 HYS-46_67 68HYS-46_68 69 HYS-46_69 70 HYS-46_70 71 HYS-46_71 72 HYS-46_72 73HYS-46_74 75 HYS-46_75 76 HYS-46_76 77 HYS-46_77 78 HYS-46_78 79HYS-46_79 80 HYS-46_80 81 HYS-46_81 82 HYS-46_82 83 HYS-46_83 84HYS-46_84 85 HYS-46_85 86 HYS-46_86 87 HYS-46_87 88 HYS-46_88 89HYS-46_89 90 HYS-46_90 91 HYS-46_91 92 HYS-46_92 93 HYS-46_93 94HYS-46_94 95 HYS-46_95 96 HYS-46_96 97 HYS-46_97 98 HYS-46_98 99HYS-46_99 100 HYS-46_100 101 HYS-46_101 102 HYS-46_102 103 HYS-46_103104 HYS-46_104 105 HYS-46_105 106 HYS-46_106 107 HYS-46_107 108HYS-46_108 109 HYS-46_109 110 HYS-46_110 111 HYS-46_111 112 HYS-46_112113 HYS-46_113 114 HYS-46_114 115 HYS-46_115 116 HYS-46_116 117HYS-46_117 118 HYS-46_118 119 HYS-46_119 120 HYS-46_120 121 HYS-46_121122 HYS-46_122 123 HYS-46_123 124 HYS-46_124 125 HYS-46_125 126HYS-46_126 127 HYS-46_127 128 HYS-46_128 129 HYS-46_129 130 HYS-46_130131 HYS-46_131 132 HYS-46_132 133 HYS-46_133 134 HYS-46_134 135HYS-46_135 136 HYS-46_136 137 HYS-46_137 138 HYS-46_138 139 HYS-46_139140 HYS-46_140 141 HYS-46_141 142 HYS-46_142 143 HYS-46_143 144HYS-46_144 145 HYS-46_145 146 HYS-46_146 147 HYS-46_147 148 HYS-46_148149 HYS-46_149 150 HYS-46_150 151 HYS-46_151 152 HYS-46_152 153HYS-46_153 154 HYS-46_154 155 HYS-46_155 156 HYS-46_156 157 HYS-46_157158 HYS-46_158 159 HYS-46_159 160 HYS-46_160 161 HYS-46_161 162HYS-46_162 163 HYS-46_163 164 HYS-46_164 165 HYS-46_165 166 HYS-46_166167 HYS-46_167 168 HYS-46_168 169 HYS-46_169 170 HYS-46_170 171HYS-46_171 172 HYS-46_172 173 HYS-46_173 174 HYS-46_174 175 HYS-46_175176 HYS-46_176 177 HYS-46_177 178 HYS-46_178 179 HYS-46_179 180HYS-46_180 181 HYS-46_181 182 HYS-46_182 183 HYS-46_183 184 HYS-46_184185 HYS-46_185 186 HYS-46_186 187 HYS-46_187 188 HYS-46_188 189HYS-46_189 190 HYS-46_190 191 HYS-46_191 192 HYS-46_192 193 HYS-46_193194 HYS-46_194 195 HYS-46_195 196 HYS-46_196 197 HYS-46_197 198HYS-46_198 199 HYS-46_199 200 HYS-46_200 201 HYS-46_201 202 HYS-46_202203 HYS-46_203 204 HYS-46_204 205 HYS-46_205 206 HYS-46_206 207HYS-46_207 208 HYS-46_208 209 HYS-46_209 210 HYS-46_210 211 HYS-46_211212 HYS-46_212 213 HYS-46_213 214 HYS-46_214 215 HYS-46_215 216HYS-46_216 217 HYS-46_217 218 HYS-46_218 219 HYS-46_219 220 HYS-46_220221 HYS-46_221 222 HYS-46_222 223 HYS-46_223 224 HYS-46_224 225HYS-46_225 226 HYS-46_226 227 HYS-46_227 228 HYS-46_228 229 HYS-46_229230 HYS-46_230 231 HYS-46_231 232 HYS-46_232 233 HYS-46_233 234HYS-46_234 235 HYS-46_235 236 HYS-46_236 237 HYS-46_237 238 HYS-46_238239 HYS-46_239 240 HYS-46_240 241 HYS-46_241 242 HYS-46_242 243HYS-46_243 244 HYS-46_244 245 HYS-46_245 246 HYS-46_246 247 HYS-46_247248 HYS-46_248 249 HYS-46_249 250 HYS-46_250 251 HYS-46_251 252HYS-46_252 253 HYS-46_253 254 HYS-46_254 255 HYS-46_255 256 HYS-46_256257 HYS-46_257 258 HYS-46_258 259 HYS-46_259 260 HYS-46_260 261HYS-46_261 262 HYS-46_262 263 HYS-46_263 264 HYS-46_264 265 HYS-46_265266 HYS-46_266 267 HYS-46_267 268 HYS-46_268 269 HYS-46_269 270HYS-46_270 271 HYS-46_271 272 HYS-46_272 273 HYS-46_273 274 HYS-46_274275 HYS-46_275 276 HYS-46_276 277 HYS-46_277 278 HYS-46_278 279HYS-46_279 280 HYS-46_280 281 HYS-46_281 282 HYS-46_282 283 HYS-46_283284 HYS-46_284 285 HYS-46_285 286 HYS-46_286 287 HYS-46_287 288HYS-46_288 289 HYS-46_289 290 HYS-46_290 291 HYS-46_291 292 HYS-46_292293 HYS-46_293 294 HYS-46_294 295 HYS-46_295 296 HYS-46_296 297HYS-46_297 198 HYS-46_298 299 HYS-46_299 300 HYS-46_300 301 HYS-46_301302 HYS-46_302 303 HYS-46_303 304 HYS-46_304 305 HYS-46_305 306HYS-46_306 307 HYS-46_307 308 HYS-46_308 309 HYS-46_309 310 HYS-46_310311 HYS-46_311 312 HYS-46_312 313 HYS-46_313 314 HYS-46_314 315HYS-46_315 316 HYS-46_316 317 HYS-46_317 318 HYS-46_318 319 HYS-46_319320 HYS-46_320 321 HYS-46_321 322 HYS-46_322 323 HYS-46_323 324HYS-46_324 325 HYS-46_325 326 HYS-46_326 327 HYS-46_327 328 HYS-46_328329 HYS-46_329 330 HYS-46_330 331 HYS-46_331 332 HYS-46_332 333HYS-46_333 334 HYS-46_334 335 HYS-46_335 336 HYS-46_336 337 HYS-46_337338 HYS-46_338 339 HYS-46_339 340 HYS-46_340 341 HYS-46_341 342HYS-46_342 343 HYS-46_343 344 HYS-46_344 345 HYS-46_345 346 HYS-46_346347 HYS-46_347 348 HYS-46_348 349 HYS-46_349 350 HYS-46_350 351HYS-46_351 352 HYS-46_352 353 HYS-46_353 354 HYS-46_354 355 HYS-46_355356 HYS-46_356 357 HYS-46_357 358 HYS-46_358 359 HYS-46_359 360HYS-46_360 361 HYS-46_361 362 HYS-46_362 363 HYS-46_363 364 HYS-46_364365 HYS-46_365 366 HYS-46_366 367 HYS-46_367 368 HYS-46_368 369HYS-46_369 370 HYS-46_370 371 HYS-46_371 372 HYS-46_372 373 HYS-46_373374 HYS-46_374 375 HYS-46_375 376 HYS-46_376 377 HYS-46_377 378HYS-46_378 379 HYS-46_379 380 HYS-46_380 381 HYS-46_381 382 HYS-46_382383 HYS-46_383 384 HYS-46_384 385 HYS-46_385 386 HYS-46_386 387HYS-46_387 388 HYS-46_388 389 HYS-46_389 390 HYS-46_390 391 HYS-46_391392 HYS-46_392 393 HYS-46_393 394 HYS-46_394 395 HYS-46_395 396HYS-46_396 397 HYS-46_397 398 HYS-46_398 399 HYS-46_399 400 HYS-46_400401 HYS-46_401 402 HYS-46_402 403 HYS-46_403 404 HYS-46_404 405 HYS-47_1406 HYS-47_2 407 HYS-47_3 408 HYS-47_4 409 HYS-47_5 410 HYS-47_6 411HYS-47_7 412 HYS-47_8 413 HYS-47_9 414 HYS-47_10 415 HYS-47_11 416HYS-47_12 417 HYS-47_13 418 HYS-52_1 419 HYS-52_2 420 HYS-52_3 421HYS-52_4 422 HYS-52_5 423 HYS-52_6 424 HYS-52_7 425 HYS-52_8 426HYS-52_9 427 HYS-52_10 428 HYS-52_11 429 HYS-52_12 430 HYS-52_13 431HYS-52_14 432 HYS-52_15 433 HYS-52_16 434 HYS-52_17 435 HYS-52_18 436HYS-52_19 437 HYS-52_20 438 HYS-52_21 439 HYS-52_22 440 HYS-52_23 441HYS-54_1 442 HYS-54_2 443 HYS-54_3 444 HYS-54_4 445 HYS-54_5 446HYS-54_6 447 HYS-54_7 448 HYS-54_8 449 HYS-54_9 450 HYS-54_10 451HYS-54_11 452 HYS-54_12 453 HYS-54_13 454 HYS-54_14 455 HYS-54_15 456HYS-54_16 457 HYS-54_17 458 HYS-54_18 459 HYS-54_19 460 HYS-54_20 461HYS-54_21 462 HYS-54_22 463 HYS-54_23 464 HYS-54_24 465 HYS-54_25 466HYS-54_26 467 HYS-54_27 468 HYS-54_28 469 HYS-54_29 470 HYS-54_30 471HYS-54_31 472 HYS-54_32 473 HYS-54_33 474 HYS-54_34 475 HYS-54_35 476HYS-54_36 477 HYS-54_37 478 HYS-54_38 479 HYS-54_39 480 HYS-54_40 481HYS-54_41 482 HYS-54_42 483 HYS-54_43 484 HYS-54_44 485 HYS-55_1 486HYS-55_2 487 HYS-55_3 488 HYS-55_4 489 HYS-55_5 490 HYS-55_6 491HYS-55_7 492 HYS-55_8 493 HYS-55_9 494 HYS-55_10 495 HYS-55_11 496HYS-55_12 497 HYS-55_13 498 HYS-55_14 499 HYS-55_15 500 HYS-55_16 501HYS-55_17 502 HYS-55_18 503 HYS-55_19 504 HYS-55_20 505 HYS-55_21 506HYS-55_22 507 HYS-55_23 508 HYS-55_24 509 HYS-55_25 510 HYS-55_26 511HYS-55_27 512 HYS-55_28 513 HYS-55_29 514 HYS-55_30 515 HYS-55_31 516HYS-55_32 517 HYS-55_33 518 HYS-55_34 519 HYS-55_35 520 HYS-55_36 521HYS-55_37 522 HYS-55_38 523 HYS-55_39 524 HYS-55_40 525 HYS-55_41 526HYS-55_42 527 HYS-55_43 528 HYS-55_44 529 HYS-55_45 530 HYS-55_46 531HYS-55_47 532 HYS-55_48 533 HYS-55_49 534 HYS-55_50 535 HYS-55_51 536HYS-55_52 537 HYS-55_53 538 HYS-55_54 539 HYS-55_55 540 HYS-55_56 547HYS-57_1 548 HYS-57_2 549 HYS-57_3 550 HYS-57_4 551 HYS-57_5 552HYS-57_6 553 HYS-57_7 554 HYS-57_8 555 HYS-57_9 556 HYS-57_10 557HYS-57_11 558 HYS-57_12 559 HYS-57_13 560 HYS-57_14 561 HYS-57_15 562HYS-57_16 563 HYS-57_17 564 HYS-57_18 565 HYS-57_19 566 HYS-57_20 567HYS-57_21 568 HYS-57_22 569 HYS-57_23 570 HYS-58_1 571 HYS-58_2 572HYS-58_3 573 HYS-58_4 574 HYS-58_5 575 HYS-58_6 576 HYS-58_7 577HYS-58_8 578 HYS-58_9 579 HYS-58_10 580 HYS-58_11 581 HYS-58_12 582HYS-58_13 583 HYS-58_14 584 HYS-58_15 585 HYS-58_16 586 HYS-58_17 587HYS-60_1 588 HYS-60_2 589 HYS-60_3 590 HYS-60_4 591 HYS-60_5 592HYS-60_6 593 HYS-60_7 594 HYS-60_8 595 HYS-60_9 596 HYS-60_10 597HYS-60_11 598 HYS-60_12 599 HYS-60_13 600 HYS-60_14 601 HYS-61_1 602HYS-61_2 603 HYS-61_3 604 HYS-61_4 605 HYS-61_5 606 HYS-61_6 607HYS-61_7 608 HYS-61_8 609 HYS-61_9 61 HYS-61_10 629 HYS-62_1 630HYS-62_2 631 HYS-62_3 632 HYS-62_4 633 HYS-62_5*HYS-46_XXX = SEQ ID NO: XXX of Attorney Docket No. HYS-46, U.S. Ser.No. 10/005,499 filed 12/03/2001, the entire disclosure of which,including sequence listing, is incorporated herein by reference.HYS-47_XXX = SEQ ID NO: XXX of Attorney Docket No. HYS-47, U.S. Ser. No.60/341,362 filed 12/17/2001, the entire disclosure of which, includingsequence listing, is incorporated herein by reference.HYS-52_XXX = SEQ ID NO: XXX of Attorney Docket No. HYS-52, U.S. Ser. No.60/379,875 filed 05/10/2002, the entire disclosure of which, includingsequence listing, is incorporated herein by reference.HYS-54_XXX = SEQ ID NO: XXX of Attorney Docket No. HYS-54, U.S. Ser. No.60/379,834 filed 05/10/2002, the entire disclosure of which, includingsequence listing, is incorporated herein by reference.HYS-55_XXX = SEQ ID NO: XXX of Attorney Docket No. HYS-55, U.S. Ser. No.60/384,450 filed 05/31/2002, the entire disclosure of which, includingsequence listing, is incorporated herein by reference.HYS-57_XXX = SEQ ID NO: XXX of Attorney Docket No. HYS-57, U.S. Ser. No.60/384,665 filed 05/31/2002, the entire disclosure of which, includingsequence listing, is incorporated herein by reference.HYS-58_XXX = SEQ ID NO: XXX of Attorney Docket No. HYS-58, U.S. Ser. No.60/389,715 filed 06/17/2002, the entire disclosure of which, includingsequence listing, is incorporated herein by reference.HYS-60_XXX = SEQ ID NO: XXX of Attorney Docket No. HYS-60, U.S. Ser. No.60/393,722 filed 07/02/2002, the entire disclosure of which, includingsequence listing, is incorporated herein by reference.HYS-61_XXX = SEQ ID NO: XXX of Attorney Docket No. HYS-61, U.S. Ser. No.60/390,531 filed 06/21/2002, the entire disclosure of which, includingsequence listing, is incorporated herein by reference.HYS-62_XXX = SEQ ID NO: XXX of Attorney Docket No. HYS-62, U.S. Ser. No.60/391,326 filed /06/24/2002, the entire disclosure of which, includingsequence listing, is incorporated herein by reference.4.1 Adiponectin-Like Polypeptides And Polynucleotides

Adipose tissue primarily serves as an energy reservoir by storing fatand is involved in regulating available energy to the body. However, ithas only recently become apparent that adipocytes synthesize and secretemany important proteins, including leptin, adipsin, complementcomponents such as C3a and properdin, tumor necrosis factor (TNF)-α,plasminogen-activator inhibitor type 1 (PAI-1), and resistin. Theseadipocyte proteins are collectively called adipocytokines (Yamauchi etal., Nature Med. 7:941-946 (2001), herein incorporated by reference).

Adiponectin (also known as adipocyte complement-related protein, Acrp30,gelatin-binding protein (GBP28), or APM1) is such an adipocytokine thatwas identified by differential display cloning of preadipocytes andadipocytes in mouse cells. In humans, it was identified as anadipocyte-specific gene. There appears to be a large family of relatedproteins that share both sequence and structural homology including C1q,human type VIII and X collagens, precerebellin, and thehibernation-regulated proteins, hib 20, hib 25, and hib 27. Adiponectin(AdipoQ) has a modular design: a cleaved amino-terminal sequence, aregion without homology to known proteins, a collagen-like region, and aC-terminal complement factor C1Q-like globular domain (Fruebis et al.,Proc. Natl. Acad. Sci. USA 98:2005-2010 (2001), herein incorporated byreference). The globular domain forms homotrimers like TNF-α, and thecollagen-like domains can further form higher order structures.

Functionally, adiponectin was found to suppress TNF-α-induced monocyteadhesion to human aortic endothelial cells (Ouchi et al., Circulation100:2473-2476 (1999), herein incorporated by reference). They alsoreported that adiponectin suppressed the increased expression of VCAM-1,ICAM-1, and E-selectin, suggesting that adiponectin may attenuate theinflammatory responses associated with atherosclerosis. More recently,authors also reported that plasma levels of adiponectin weresignificantly lower in patients with coronary artery disease than in ageand body mass index-matched normal subjects (Ouchi et al., Circulation102:1296-1301 (2000), herein incorporated by reference). It was furthershown that adiponectin suppressed TNF-α-induced nuclear factor Kappa B(NF-κB) activation accompanied by cAMP accumulation. Adiponectin alsoinhibited myelomonocytic progenitor cell proliferation, at least in partdue to apoptotic mechanisms in hematopoietic colony formation assays. Inmacrophages, adiponectin suppressed the expression of class A macrophagescavenger receptors (MSR) and altered cholesterol metabolism. Inparticular, adiponectin reduced intracellular cholesteryl ester contentof the macrophages (Ouchi et al., Circulation 103:1057-63 (2001), hereinincorporated by reference). The findings suggested that adiponectinprotein suppressed the transformation of macrophages to foam cells.

Insulin resistance induced by high-fat diet and associated with obesityis a major risk factor for diabetes and cardiovascular diseases. It hasbeen shown that adipocytokines play a crucial role in these processes.TNF-α overproduced in adipose tissue contributes to insulin resistance.Leptin, another adipocytokine, which contributes to the regulation offood intake and energy expenditure, also affects insulin sensitivity andmay lead to hypertension. Similarly, serum adiponectin concentrationsare decreased in homozygous obese (ob/ob) mice, obese humans, diabeticpatients, and patients with coronary artery diseases (Hotta et al.Arterioscler. Thromb. Vasc. Biol. 20:1595-1599 (2000), hereinincorporated by reference).

In mouse models, it was shown that acute treatment with aproteolytically generated globular domain of Acrp30 (gAcrp30) could leadto altered lipid metabolism. In particular, the gAcrp30 reduced plasmafatty acid levels caused by administration of a high-fat test meal(Freubis et al., Proc. Natl. Acad. Sci. USA 98:2005-2010 (2001), hereinincorporated by reference). This effect was in part due to increasedfatty acid oxidation by muscle. Low doses of gAcrp30 given to mice thatwere on high-fat/sucrose diet caused profound and sustainable weightreduction without affecting food intake. These data indicated thatadiponectin as well as other adiponectin family members may be involvedin energy homeostasis and their dysregulation may lead to pathologicalconditions.

Recently, Yamauchi et al. showed that decreased expression ofadiponectin correlates with insulin resistance in mouse models ofaltered insulin sensitivity (Yamauchi et al., Nature Med. 7:941-946(2001), herein incorporated by reference). Adiponectin decreased thelevels of triglycerides in muscle and liver in obese mice. These effectswere due to increased fatty acid combustion and energy dissipation inmuscle. The authors further showed that insulin resistance wascompletely reversed in lipoatrophic mice by administering combination ofphysiological doses of adiponectin and leptin, but only partially witheither adiponectin or leptin alone.

The role of adiponectin was further studied in the adiponectin knock-out(KO) mice by Matsuda et al. (J. Biol. Chem. 277:37487-37491 (2002)) andKubota et al. (J. Biol. Chem. 277:25863-25866 (2002), both hereinincorporated by reference). The adiponectin-deficient mice in each studyshowed severe neointimal thickening and increased proliferation ofvascular smooth muscle cells in mechanically injured arteries.Adenovirus-mediated supplement of adiponectin attenuated the neotintimalproliferation, suggesting that adiponectin plays a direct role inneointimal thickening of arteries, a key feature of the restenosisphenomenon observed after balloon angioplasty. In cultured smooth musclecells, adiponectin attenuated DNA synthesis induced a variety of growthfactors such as PDGF, HB-EGF, bFGF and EGF and cell proliferation andmigration induced by HB-EGF. In cultured endothelial cells, adiponectinattenuated HB-EGF expression stimulated by TNFα (Matsuda et al., J.Biol. Chem. 277:37487-37491 (2002), herein incorporated by reference).Kubota et al. further showed that the levels of FFAs, triglycerides andtotal cholesterol of adipoenctin-deficient mice were significantlyelevated indicating that the lipid metabolism of these mice was severelydisrupted and the mice were hyperlipidemic (Kubota et al., J. Biol.Chem. 277:25863-25866 (2002), herein incorporated by reference).Adiponectin therefore has antiatherogenic properties.

In a separate study of adiponectin-KO mice, Maeda et al found that therewas delayed clearance of FFA in plasma, low levels of fatty acidtransport protein 1 (FATP1) mRNA in muscle, high levels of TNFα mRNA inadipose tissue and high plasma TNFα concentrations. These KO miceexhibited severe diet-induced insulin resistence with reducedinsulin-receptor substrate 1 (IRS-1)-associated phosphatidyl inositol 3(PI3)-kinase activity in the muscles. Adenovirus-mediated adiponectinexpression in the KO mice reversed the increase of adipose TNFα mRNA andthe diet-induced insulin resistance. In cultured myocytes, TNFαdecreased FATP1 mRNA, IRS1-associated PI3-kinase activity and glucoseuptake whereas adiponectin increased these parameters supporting thesimilar observations in mice (Maeda et al., Nature Med. 8:731-737,(2002), herein incorporated by reference).

Hotta et al have shown that plasma levels of adiponectin are decreasedin Type 2 diabetes patients with coronary artery disease (CAD)complications and may cause the develoment of insulin resistance inthese patients. In addition, the plasma adiponectin levels independentlynegatively correlated with serum triglyceridemia levels suggestingdecreased adiponectin is associated with hypertriglyceridemia which isknown to play a significant role in the deveopment of atherosclerosis.In addition, sex differences were observed in adiponectin concentrationsin the diabetic subjects without CAD with higher levels in clinicallynormal women as well as in diabetic women suggesting that sex hormonesincluding estrogen, progesterone and androgen may affect plasmaadiponectin levels (Hotta et al., Arterioscler. Thromb. Vasc. Biol.20:1595-1599 (2000), herein incorporated by reference). The plasmalevels of adiponectin are also reduced in cardiovascular patients withend stage renal disease and the incidence of cardiovascular death ishigher in renal failure patients with low plasma adiponectins comparedwith those with higher plasma adiponectin levels (Zoccali et al., J AmSoc Nephrol. 13:134-41 (2002), herein incorporated by reference). Thesedata clearly show that adiponectin is involved in metabolic disordersincluding diabetes cardiovascular disease with and without renalcomplications.

Based on these studies and others, therapeutics that increase plasmaadiponectin should be useful in preventing metabolic disorders,diabetes, cardiovascular and other related disorders such asatherogenesis, hypertriglyceridemia, vascular stenosis afterangioplasty. Thus, the adiponectin-like polypeptides and polynucleotidesof the invention may be used to treat obesity, diabetes, lipoatrophy,coronary artery diseases, atherosclerosis, and other obesity anddiabetes-related cardiovascular pathologies. Adiponectin-likepolypeptides and polynucleotides of the invention may also be used intreatment of autoimmune diseases and inflammation, to modulate immuneresponses, and to treat transplant patients. Adiponectin-likepolypetides may also be used in the treatment of tumors such as solidtumors and leukemia.

Thirteen exemplary adiponectin-like sequences of the invention aredescribed below: amino acid SEQ ID NO: 5 (and encoding nucleotidesequence SEQ ID NO: 4), amino aicid SEQ ID NO: 15 (and encodingnucleotide sequence SEQ ID NO: 14), amino acid SEQ ID NO: 28 (andencoding nucleotide SEQ ID NO: 27), amino acid SEQ ID NO: 160 (andencoding nucleotide sequence 159), amino acid SEQ ID NO: 186 (andencoding nucleotide sequence SEQ ID NO: 185), amino acid SEQ ID NO: 215(and encoding nucleotide sequence SEQ ID NO: 214), amino acid sequenceSEQ ID NO: 241 (and encoding nucleotide sequence SEQ ID NO: 240), aminoacid SEQ ID NO: 272 (and encoding nucleotide sequence SEQ ID NO: 271),amino acid SEQ ID NO: 302 (and encoding nucleotide sequence SEQ ID NO:301), amino acid SEQ ID NO: 323 (and encoding nucleotide sequence SEQ IDNO: 322), amino acid SEQ ID NO: 348 (and encoding nucleotide sequenceSEQ ID NO: 347), amino acid SEQ ID NO: 355 (and encoding nucleotidesequence SEQ ID NO: 354), and amino acid SEQ ID NO: 378 (and encodingnucleotide sequence SEQ ID NO: 377).

The first adiponectin-like polypeptide of SEQ ID NO: 5 is anapproximately 800-amino acid protein with a predicted molecular mass ofapproximately 90-kDa unglycosylated. The initial methionine starts atposition 511 of SEQ ID NO: 4 and the putative stop codon begins atpositions 2911 of SEQ ID NO: 4. Protein database searches with theBLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altschul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 5 is homologous toadiponectin. Using the Pfam software program (Sonnhammer et al., NucleicAcids Res., 26:320-322 (1998) herein incorporated by reference),adiponectin-like polypeptide of SEQ ID NO: 5 revealed its structuralhomology to C1q domain. Further description of the Pfam models can befound at http://pfam.wustl.edu/.

FIG. 1 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 5 and adiponectin SEQ ID NO: 402(Hotta et al., Diabetes 50:1126-1133 (2001)), indicating that the twosequences share 49% similarity over 136 amino acid residues and 30%identity over the same 136 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G-Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

FIG. 2 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 5 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1), indicating that the two sequencesshare 49% similarity over 136 amino acid residues and 30% identity overthe same 136 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol. 6:219-235 (1999), herein incorporated byreference), adiponectin-like polypeptide of SEQ ID NO: 5 was determinedto have following eMATRIX domain hits. The results describe:corresponding SEQ ID NO: in sequence listing, e-value, subtype,Accession number, name, position of the domain in the full-lengthprotein, and the amino acid sequence and are displayed in Table 2 below,wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y'Tyrosine.TABLE 2 SEQ Amino acid ID Accession sequence (start NO; e−value SubtypeNo. Name and end position) 7 9.294e−19 18.26 BL01113B C1q domainPIVFDLLLNNLGETFDLQ proteins LGRFNCPVNGTYVFIFHM (689-725) 8 8.235e−1215.60 PR00007C Complement ETASNHAILQLFQGDQIW C1Q domain LRLH (757-779)signature 9 4.857e−11 13.18 BL01113C C1q domain ETASNHAILQLFQGDQIWproteins LR (757-777) 10 1.250e−10 9.64 PR00007D Complement KYSTFSGYLLYC1Q domain (788-799) signature 11 2.161e−10 7.47 BL01113D C1q domainSTFSGYLLYQ proteins (790-800) 12 7.107e−10 14.16 PR00007B ComplementFNCPVNGTYVFIFHMLKL C1Q domain AV (710-730) signature 13 7.517e−10 19.33PR00007A Complement PGTLDQPIVFDLLLNNLG C1Q domain ETFDLQLGR signature(683-710)

The second adiponectin-like polypeptide of SEQ ID NO: 15 is anapproximately 710-amino acid protein with a predicted molecular mass ofapproximately 80-kDa unglycosylated. The initial methionine starts atposition 511 of SEQ ID NO: 14 and the putative stop codon begins atpositions 2641 of SEQ ID NO: 14. Protein database searches with theBLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altschul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 15 is homologous toadiponectin. Using the Pfam software program (Sonnhammer et al., NucleicAcids Res., 26:320-322 (1998) herein incorporated by reference),adiponectin-like polypeptide of SEQ ID NO: 15 revealed its structuralhomology to C1q domain. Further description of the Pfam models can befound at http://pfam.wustl.edu/.

FIG. 3 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 15 and adiponectin SEQ ID NO:402 (Hotta et al., Diabetes 50:1126-1133 (2001)), indicating that thetwo sequences share 47% similarity over 136 amino acid residues and 29%identity over the same 136 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

FIG. 4 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 15 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B 1), indicating that the two sequencesshare 48% similarity over 136 amino acid residues and 29% identity overthe same 136 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), adiponectin-like polypeptide of SEQ ID NO: 15 was determinedto have following eMATRIX domain hits. The results describe:corresponding SEQ ID NO: in sequence listing, e-value, subtype,Accession number, name, position of the domain in the full-lengthprotein, and the amino acid sequence and are shown in Table 3 below,wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.TABLE 3 SEQ Amino acid ID Accession sequence (start NO; e−value SubtypeNo. Name and end position) 17 3.813e−14 18.26 BL01113B C1q domainPYGVDLLLNNLGETFDL proteins QLGRFNCPVNGTYVFIFH M (599-635) 18 8.235e−1215.60 PR00007C Complement ETASNHAILQLFQGDQIW C1Q domain LRLH (667-689)signature 19 4.857e−11 13.18 BL01113C C1q domain ETASNHAILQLFQGDQIWproteins LR (667-687) 20 1.250e−10 9.64 PR00007D Complement KYSTFSGYLLYQC1Q domain (698-709) signature 21 2.161e−10 7.47 BL01113D C1q domainSTFSGYLLYQ proteins (700-710) 22 7.107e-10 14.16 PR00007B ComplementFNCPVNGTYVFIFHMLKL C1Q domain AV (620-640) signature

The third adiponectin-like polypeptide of SEQ ID NO: 28 is anapproximately 744-amino acid protein with a predicted molecular mass ofapproximately 83-kDa unglycosylated. The initial methionine starts atposition 235 of SEQ ID NO: 27 and the putative stop codon begins atpositions 2467 of SEQ ID NO: 27. Protein database searches with theBLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altshul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 28 is homologous toadiponectin. Using the Pfam software program (Sonnhammer et al., NucleicAcids Res., 26:320-322 (1998) herein incorporated by reference),adiponectin-like polypeptide of SEQ ID NO: 28 revealed its structuralhomology to C1q, and collagen domains. Further description of the Pfammodels can be found at http://pfam.wustl.edu/.

FIG. 5 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 28 and adiponectin SEQ ID NO:402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the twosequences share 55% similarity over 225 amino acid residues and 37%identity over the same 225 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

FIG. 6 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 28 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1), indicating that the two sequencesshare 54% similarity over 236 amino acid residues and 36% identity overthe same 236 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), adiponectin-like polypeptide of SEQ ID NO: 28 was determinedto have following eMATRIX domain hits. The results describe:corresponding SEQ ID NO: in sequence listing, e-value, subtype,Accession number, name, position of the domain in the full-lengthprotein, and the amino acid sequence and are shown in Table 4 below,wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.TABLE 4 SEQ Amino acid ID Accession sequence (start NO; e−value SubtypeNo. Name and end position) 32 5.500e-35 18.26 BL01113B C1q domainPVKFNKLLYNGRQNY proteins NPQTGIFTCEVPGVYY FAYHV (632-668) 33 8.615e-2314.16 PR00007B Complement FTCEVPGVYYFAYHV C1q domain HCKGG signature(653-673) 34 6.192e-22 19.33 PR00007A Complement FPPVGAPVKFNKLLY C1qdomain NGRQNYNPQTGI signature (626-653) 35 5.846e−19 15.60 PR00007CComplement DQASGSAVLLLRPGD C1q domain RVFLQ (698-720) signature 366.700e−17 13.18 BL01113C C1q domain DQASGSAVLLLRPGD proteins RVFLQ(698-718) 37 6.885e−17 17.99 BL01113A C1q domain PGPHGLPGIGKPGGPGproteins LPGQPGPKGDR (199-226) 38 9.357e−15 20.42 BL00420A Speractreceptor GPPGAIGFPGPKGEGG repeat proteins IVGPQGPPGPKGE domain proteins(402-431) 39 4.545e−14 17.99 BL01113A C1q domain GPPGIPGIGGPSGPIGPproteins PGIPGPKGEP (495-522) 40 8.636e−14 17.99 BL01113A C1q domainGPPGEPGLPGIPGPMG proteins PPGAIGFPGPK (387-414) 41 1.486e−13 17.99BL01113A C1q domain GVPGLLGPKGEPGIPG proteins DQGLQGPPGIP (474-501) 421.730e−13 17.99 BL01113A C1q domain GKPGMPGMPGKPGA proteinsMGMPGAKGEIGQK (158-185) 43 3.647e−13 9.64 PR00007D ComplementVHSSFSGYLLY C1q domain (732-743) signature 44 4.162e−13 17.99 BL01113AC1q domain GGPGLPGQPGPKGDR proteins GPKGLPGPQGLR (211-238) 45 5.408e−1320.42 BL00420A Speract receptor GKPGMPGMPGKPGA repeat proteinsMGMPGAKGEIGQKG domain proteins E (158-187) 46 6.838e−13 17.99 BL01113AC1q domain GIPGQPGFPGGKGEQ proteins GLPGLPGPPGLP (322-349) 47 6.838e−1317.99 BL01113A C1q domain GAPGIGGPPGEPGLPG proteins IPGPMGPPGAI(381-408) 48 7.081e−13 17.99 BL01113A C1q domain GKPGQDGIPGQPGFPGproteins GKGEQGLPGLP (316-343) 49 7.245e−13 20.42 BL00420A Speractreceptor GFPGKPGFLGEVGPPG repeat proteins MRGFPGPIGPKGE domain proteins(435-464) 50 8.541e−13 17.99 BL01113A C1q domain GPPGIPGPKGEPGLPGproteins PPGFPGIGKPG (510-537) 51 9.027e−13 17.99 BL01113A C1q domainGMPGAPGVKGPPGM proteins HGPPGPVGLPGVG (246-273) 52 9.027e−13 17.99BL01113A C1q domain GFPGPQGPLGKPGAP proteins GEPGPQGPIGVP (278-305) 531.231e−12 17.99 BL01113A C1q domain GPPGKPGALGPQGQP proteinsGLPGPPGPPGPP (542-569) 54 2.154e−12 17.99 BL01113A C1q domainGPSGPIGPPGIPGPKGE proteins PGLPGPPGFP (504-531) 55 2.615e−12 17.99BL01113A C1q domain GLPGIPGPMGPPGAIG proteins FPGPKGEGGIV (393-420) 564.231e−12 17.99 BL01113A C1q domain GKPGALGPQGQPGLP proteinsGPPGPPGPPGPP (545-572) 57 5.154e−12 20.42 BL00420A Speract receptorGPPGEPGLPGIPGPMG repeat proteins PPGAIGFPGPKGE domain proteins (387-416)58 5.327e−12 20.42 BL00420A Speract receptor GPIGPKGEHGQKGVP repeatproteins GLPGVPGLLGPKGE domain proteins (456-485) 59 7.462e−12 17.99BL01113A C1q domain PGIGKPGGPGLPGQPG proteins PKGDRGPKGLP (205-232) 608.385e−12 17.99 BL01113A C1q domain GIGGPSGPIGPPGIPGP proteinsKGEPGLPGPP (501-528) 61 8.846e−12 17.99 BL01113A C1q domainGPPGMRGFPGPIGPKG proteins EHGQKGVPGLP (447-474) 62 1.000e−11 7.47BL01113D C1q domain SSFSGYLLYP proteins (734-744) 63 1.818e−11 17.99BL01113A C1q domain GKPGGPGLPGQPGPK proteins GDRGPKGLPGPQ (208-235) 644.764e−11 20.42 BL00420A Speract receptor GEPGLPGIPGPMGPPG repeatproteins AIGFPGPKGEGGI domain proteins (390-419) 65 5.418e−11 20.42BL00420A Speract receptor PGIGKPGFPGPKGDRG repeat proteins MGGVPGALGPRGEdomain proteins (348-377) 66 5.500e−11 17.99 BL01113A C1q domainGPQGPPGPKGEPGLQ proteins GFPGKPGFLGEV (420-447) 67 5.705e−11 17.99BL01113A C1q domain PGPQGYPGVGKPGMP proteins GMPGKPGAMGMP (149-176) 686.114e−11 17.99 BL01113A C1q domain GIPGIGGPSGPIGPPGIP proteinsGPKGEPGLP (498-525) 69 6.318e−11 17.99 BL01113A C1q domainGPRGEKGPIGAPGIGG proteins PPGEPGLPGIP (372-399) 70 6.891e−11 20.42BL00420A Speract receptor GKPGFLGEVGPPGMR repeat proteins GFPGPIGPKGEHGQdomain proteins (438-467) 71 7.545e−11 17.99 BL01113A C1q domainGEPGPQGPIGVPGVQ proteins GPPGIPGIGKPG (293-320) 72 8.773e−11 17.99BL01113A C1q domain GIGGPPGEPGLPGIPGP proteins MGPPGAIGFP (384-411) 739.386e−11 17.99 BL01113A C1q domain GKPGAPGEPGPQGPIG proteinsVPGVQGPPGIP (287-314) 74 9.795e-11 17.99 BL01113A C1q domainGLPGQPGPKGDRGPK proteins GLPGPQGLRGPK (214-241) 75 1.000e−10 17.99BL01113A C1q domain GVPGLPGVPGLLGPK proteins GEPGIPGDQGLQ (468-495) 761.574e−10 17.99 BL01113A C1q domain GKPGFLGEVGPPGMR proteinsGFPGPIGPKGEH (438-465) 77 1.766e−10 17.99 BL01113A C1q domainGFPGPIGPKGEHGQK proteins GVPGLPGVPGLL (453-480) 78 2.149e−10 17.99BL01113A C1q domain QGPPGIPGIGKPGQDG proteins IPGQPGFPGGK (307-334) 792.149e−10 17.99 BL01113A C1q domain PGPPGFPGIGKPGVAG proteinsLHGPPGKPGAL (524-551) 80 2.532e−10 17.99 BL01113A C1q domainGQDGIPGQPGFPGGK proteins GEQGLPGLPGPP (319-346) 81 2.532e−10 17.99BL01113A C1q domain GPIGAPGIGGPPGEPG proteins LPGIPGPMGPP (378-405) 822.723e−10 17.99 BL01113A C1q domain GPMGPPGAIGFPGPKG proteinsEGGIVGPQGPP (399-426) 83 2.918e−10 20.42 BL00420A Speract receptorGPIGAPGIGGPPGEPG repeat proteins LPGIPGPMGPPGA domain proteins (378-407)84 3.489e−10 17.99 BL01113A C1q domain GPLGKPGAPGEPGPQ proteinsGPIGVPGVQGPP (284-311) 85 3.681e−10 17.99 BL01113A C1q domainPGVGKPGMPGMPGKP proteins GAMGMPGAKGEI (155-182) 86 3.681e−10 17.99BL01113A C1q domain GMPGMPGKPGAMGM proteins PGAKGEIGQKGEI (161-188) 873.872e−10 17.99 BL01113A C1q domain GEPGLQGFPGKPGFL proteinsGEVGPPGMRGFP (429-456) 88 4.255e−10 17.99 BL01113A C1q domainGQPGLPGPPGPPGPPG proteins PPAVMPPTPPP (554-581) 89 4.447e−10 17.99BL01113A C1q domain GLPGVPGLLGPKGEP proteins GIPGDQGLQGPP (471-498) 904.830e−10 17.99 BL01113A C1q domain GLLGPKGEPGIPGDQG proteinsLQGPPGIPGIG (477-504) 91 5.787e−10 17.99 BL01113A C1q domainGFPGGKGEQGLPGLP proteins GPPGLPGIGKPG (328-355) 92 5.787e−10 17.99BL01113A C1q domain GFPGKPGFLGEVGPPG proteins MRGFPGPIGPK (435-462) 935.979e−10 17.99 BL01113A C1q domain GPQGQPGLPGPPGPPG proteinsPPGPPAVMPPT (551-578) 94 6.016e−10 20.42 BL00420A Speract receptorGIPGQPGFPGGKGEQ repeat proteins GLPGLPGPPGLPGI domain proteins (322-351)95 6.170e−10 17.99 BL01113A C1q domain PGIGKPGQDGIPGQPG proteinsFPGGKGEQGLP (313-340) 96 6.170e−10 17.99 BL01113A C1q domainGLHGPPGKPGALGPQ proteins GQPGLPGPPGPP (539-566) 97 6.459e−10 20.42BL00420A Speract receptor QGYPGVGKPGMPGM repeat proteins PGKPGAMGMPGAKGdomain proteins E (152-181) 98 6.553e−10 17.99 BL01113A C1q domainGQKGVPGLPGVPGLL proteins GPKGEPGIPGDQ (465-492) 99 6.553e−10 17.99BL01113A C1q domain GIPGPKGEPGLPGPPG proteins FPGIGKPGVAG (513-540) 1006.902e−10 20.42 BL00420A Speract receptor GMPGMPGKPGAMGM repeat proteinsPGAKGEIGQKGEIGP domain proteins (161-190) 101 6.936e−10 17.99 BL01113AC1q domain GALGPQGQPGLPGPP proteins GPPGPPGPPAVM (548-575) 102 7.511e−1017.99 BL01113A C1q domain GVAGLHGPPGKPGAL proteins GPQGQPGLPGPP(536-563) 103 7.702e−10 17.99 BL01113A C1q domain PGPPGLPGIGKPGFPGproteins PKGDRGMGGVP (342-369) 104 7.787e−10 20.42 BL00420A Speractreceptor GPPGKPGALGPQGQP repeat proteins GLPGPPGPPGPPGP domain proteins(542-571) 105 8.277e−10 17.99 BL01113A C1q domain GQPGFPGGKGEQGLPproteins GLPGPPGLPGIG (325-352) 106 8.672e−10 20.42 BL00420A Speractreceptor GKPGFPGPKGDRGMG repeat proteins GVPGALGPRGEKGP domain proteins(351-380) 107 9.071e−10 0.00 PR00049D Wilm+S Tumor GPPGPPAVMPPTPPPprotein (566-581) signature 108 9.115e−10 20.42 BL00420A Speractreceptor PGVGKPGMPGMPGKP repeat proteins GAMGMPGAKGEIGQ domain proteins(155-184) 109 9.234e−10 17.99 BL01113A C1q domain GPKGEHGQKGVPGLPproteins GVPGLLGPKGEP (459-486) 110 9.426e−10 17.99 BL01113A C1q domainGPQGPLGKPGAPGEP proteins GPQGPIGVPGVQ (281-308) 111 9.518e−10 19.43DM00215 Proline-rich LGPQGQPGLPGPPGPP protein 3 GPPGPPAVMPPTPPPQ G(550-583) 112 1.000e−09 17.99 BL01113A C1q domain GMPGKPGAMGMPGAproteins KGEIGQKGEIGPM (164-191) 113 1.173e−09 17.99 BL01113A C1q domainGVPGALGPRGEKGPI proteins GAPGIGGPPGEP (366-393) 114 1.692e−09 17.99BL01113A C1q domain GQPGPKGDRGPKGLP proteins GPQGLRGPKGDK (217-244) 1151.692e−09 17.99 BL01113A C1q domain GPIGPPGIPGPKGEPGL proteinsPGPPGFPGIG (507-534) 116 1.692e−09 17.99 BL01113A C1q domainGKPGVAGLHGPPGKP proteins GALGPQGQPGLP (533-560) 117 1.865e−09 17.99BL01113A C1q domain GEPGLPGIPGPMGPPG proteins AIGFPGPKGEG (390-417) 1182.212e−09 17.99 BL01113A C1q domain PGPVGLPGVGKPGVT proteinsGFPGPQGPLGKP (263-290) 119 2.385e−09 17.99 BL01113A C1q domainGAPGEPGPQGPIGVPG proteins VQGPPGIPGIG (290-317) 120 2.731e−09 17.99BL01113A C1q domain PGVGKPGVTGFPGPQ proteins GPLGKPGAPGEP (269-296) 1212.938e−09 20.42 BL00420A Speract receptor GIPGDQGLQGPPGIPGI repeatproteins GGPSGPIGPPGI domain proteins (486-515) 122 3.423e−09 17.99BL01113A C1q domain GEGGIVGPQGPPGPK proteins GEPGLQGFPGKP (414-441) 1233.492e−09 20.42 BL00420A Speract receptor GLQGPPGIPGIGGPSG repeatproteins PIGPPGIPGPKGE domain proteins (492-521) 124 3.797e−09 13.84DM00250B kw Annexin GQPGLPGPPGPPGPPG antigen proline PPAVMPPT (554-578)tumor 125 4.288e−09 17.99 BL01113A C1q domain GPPGPKGEPGLQGFPG proteinsKPGFLGEVGPP (423-450) 126 4.288e−09 17.99 BL01113A C1q domainGIPGDQGLQGPPGIPGI proteins GGPSGPIGPP (486-513) 127 4.323e−09 20.42BL00420A Speract receptor GEPGLQGFPGKPGFL repeat proteins GEVGPPGMRGFPGPdomain proteins (429-458) 128 5.073e−09 4.29 BL00415N SynapsinsPPGKPGALGPQGQPG proteins LPGPPGPPGPPGPPAV MPPTPPPQGEYLP (543-587) 1295.401e−09 4.29 BL00415N Synapsins MPGAPGVKGPPGMH proteinsGPPGPVGLPGVGKPG VTGFPGPQGPLGKPG (247-291) 130 5.467e−09 4.29 BL00415NSynapsins PQGPLGKPGAPGEPGP proteins QGPIGVPGVQGPPGIP GIGKPGQDGIPG(282-326) 131 5.569e−09 20.42 BL00420A Speract receptorGPPGIPGIGGPSGPIGP repeat proteins PGIPGPKGEPGL domain proteins (495-524)132 5.821e−09 15.53 PD01234B Protein nuclear PGPPGPPGPPAVMPPTbromodomain PP (562-580) trans. 133 6.019e−09 17.99 BL01113A C1q domainGEVGPPGMRGFPGPIG proteins PKGEHGQKGVP (444-471) 134 6.019e−09 17.99BL01113A C1q domain GEHGQKGVPGLPGVP proteins GLLGPKGEPGIP (462-489) 1356.186e−09 0.00 PR00049D Wilm's Tumor GLPGPPGPPGPPGPP protein (557-572)signature 136 6.365e−09 17.99 BL01113A C1q domain GLPGPPGPPGPPGPPAproteins VMPPTPPPQGE (557-584) 137 6.365e−09 17.99 BL01113A C1q domainGPPGPPGPPGPPAVMP proteins PTPPPQGEYLP (560-587) 138 6.954e−09 20.42BL00420A Speract receptor GGPGLPGQPGPKGDR repeat proteins GPKGLPGPQGLRGPdomain proteins (211-240) 139 7.404e−09 17.99 BL01113A C1q domainGMPGAKGEIGQKGEI proteins GPMGIPGPQGPP (173-200) 140 7.621e−09 4.49BL00291A Prion protein PGIGKPGGPGLPGQPG PKGDRGPKGLPGPQG LRGP (205-240)141 7.923e−09 17.99 BL01113A C1q domain GKPGVTGFPGPQGPL proteinsGKPGAPGEPGPQ (272-299) 142 8.477e−09 20.42 BL00420A Speract receptorGPKGEHGQKGVPGLP repeat proteins GVPGLLGPKGEPGI domain (459-488)proteins. 143 8.615e−09 20.42 BL00420A Speract receptor GQPGFPGGKGEQGLPrepeat proteins GLPGPPGLPGIGKP domain proteins (325-354) 144 8.615e−0917.99 BL01113A C1q domain GAIGFPGPKGEGGIVG proteins PQGPPGPKGEP(405-432) 145 8.752e−09 4.29 BL00415N Synapsins PKGEPGLPGPPGFPGIproteins GKPGVAGLHGPPGKP GALGPQGQGLPG (517-561) 146 8.754e−09 20.42BL00420A Speract receptor GAPGIGGPPGEPGLPG repeat proteins IPGPMGPPGAIGFdomain proteins (381-410) 147 9.169e−09 20.42 BL00420A Speract receptorGLPGQPGPKGDRGPK repeat proteins GLPGPQGLRGPKGD domain proteins (214-243)148 9.169e−09 20.42 BL00420A Speract receptor GMGGVPGALGPRGE repeatproteins KGPIGAPGIGGPPGE domain proteins (363-392) 149 9.308e−09 20.42BL00420A Speract receptor GPIGPPGIPGPKGEPGL repeat proteins PGPPGFPGIGKPdomain proteins (507-536) 150 9.542e−09 0.00 PR00049D Wilm's TumorPGPPGPPAVMPPTPP protein (565-580) signature 151 9.585e−09 20.42 BL00420ASperact receptor GKPGVTGFPGPQGPL repeat proteins GKPGAPGEPGPQGP domainproteins (272-301) 152 9.827e−09 17.99 BL01113A C1q domainGKPGAMGMPGAKGEI proteins GQKGEIGPMGIP (167-194) 153 1.000e−08 20.42BL00420A Speract receptor GFLGEVGPPGMRGFP repeat proteins GPIGPKGEHGQKGVdomain proteins (441-470) 154 1.000e−08 17.99 BL01113A C1q domainSLRGEQGPRGEPGPR proteins GPPGPPGLPGHG (115-142) 155 1.000e−08 17.99BL01113A C1q domain GPKGEPGLQGFPGKP proteins GFLGEVGPPGMR (426-453)

A predicted approximately twenty seven-residue signal peptide is encodedfrom approximately residue 1 to residue 27 of SEQ ID NO: 28 (SEQ ID NO:30). The extracellular portion is useful on its own. This can beconfirmed by expression in mammalian cells and sequencing of the cleavedproduct. The signal peptide region was predicted using the NeuralNetwork SignalP V1.1 program (Nielsen et al, Int. J. Neural Syst.8:581-599 (1997)). One of skill in the art will recognize that theactual cleavage site may be different than that predicted by thecomputer program. SEQ ID NO: 31 is the resulting peptide when the signalpeptide is removed from SEQ ID NO: 28.

The fourth adiponectin-like polypeptide of SEQ ID NO: 160 is anapproximately 289-amino acid protein with a predicted molecular mass ofapproximately 32-kDa unglycosylated. The initial methionine starts atposition 80 of SEQ ID NO: 159 and the putative stop codon begins atpositions 947 of SEQ ID NO: 159. Protein database searches with theBLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altschul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 160 is homologous toadiponectin. Using the Pfam software program (Sonnhammer et al., NucleicAcids Res., 26:320-322 (1998) herein incorporated by reference),adiponectin-like polypeptide of SEQ ID NO: 160 revealed its structuralhomology to C1q and collagen domains. Further description of the Pfammodels can be found at http://pfam.wustl.edu/.

FIG. 7 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 160 and adiponectin SEQ ID NO:402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the twosequences share 58% similarity over 228 amino acid residues and 40%identity over the same 228 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

FIG. 8 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 160 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1), indicating that the two sequencesshare 56% similarity over 238 amino acid residues and 39% identity overthe same 238 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), adiponectin-like polypeptide of SEQ ID NO: 160 wasdetermined to have following eMATRIX domain hits. The results describe:corresponding SEQ ID NO: in sequence listing, e-value, subtype,Accession number, name, position of the domain in the full-lengthprotein, and the amino acid sequence and are shown in Table 5 below,wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.TABLE 5 SEQ Amino acid ID Accession sequence (start NO; e−value SubtypeNo. Name and end position) 164 1.581e-29 18.26 BL01113B C1q domainPIIFNKVLFNEGEHYN proteins PATGKFICAFPGIYYFS YDI (164-200) 165 1.000e−1619.33 PR00007A Complement YPEERLPIIFNKVLFNE C1q domain GEHYNPATGKsignature (158-185) 166 3.077e−15 13.18 BL01113C C1q domainDVASGSTVIYLQPEDE proteins VWLE (229-249) 167 8.200e−15 15.60 PR00007CComplement DVASGSTVIYLQPEDE C1q domain VWLEIF (229-251) signature 1685.846e−14 14.16 PR00007B Complement FICAFPGYYFSYDITL C1q domain ANK(185-205) signature 169 1.243e−13 17.99 BL01113A C1q domainGSPGPHGRIGLPGRDG proteins RDGRKGEKGEK (50-77) 170 6.108e−13 17.99BL01113A C1q domain SIPGLPGPPGPPGANG proteins SPGPHGRIGLP (35-62) 1713.077e−12 17.99 BL01113A C1q domain GPPGPPGANGSPGPH proteinsGRIGLPGRDGRD (41-68) 172 5.154e−12 20.42 BL00420A Speract receptorGPPGANGSPGPHGRIG repeat proteins LPGRDGRDGRKGE domain proteins (44-73)173 1.655e−11 20.42 BL00420A Speract receptor GPLGLAGEKGDQGET repeatproteins GKKGPIGPEGEKGE domain proteins (86-115) 174 1.574e−10 17.99BL01113A C1q domain GLPGPPGPPGANGSPG proteins PHGRIGLPGRD (38-65) 1752.328e−10 20.42 BL00420A Speract receptor GKKGPIGPEGEKGEV repeatproteins GPIGPPGPKGDRGE domain proteins (101-130) 176 5.250e−10 9.64PR00007D Complement ADSLFSGFLLY C1q domain (264-275) signature 1779.617e−10 17.99 BL01113A C1q domain GPPGANGSPGPHGRIG proteinsLPGRDGRDGRK (44-71) 178 4.185e−09 20.42 BL00420A Speract receptorGANGSPGPHGRIGLPG repeat proteins RDGRDGRKGEKGE domain proteins (47-76)179 7.577e−09 17.99 BL01113A C1q domain GLPGRDGRDGRKGEK proteinsGEKGTAGLRGKT (59-86) 180 7.577e−09 17.99 BL01113A C1q domainGEKGEVGPIGPPGPKG proteins DRGEQGDPGL (110-137) 181 9.031e−09 20.42BL00420A Speract receptor GSPGPHGRIGLPGRDG repeat proteins RDGRKGEKGEKGTdomain proteins (50-79)

A predicted approximately sixteen-residue signal peptide is encoded fromapproximately residue 1 to residue 16 of SEQ ID NO: 160 (SEQ ID NO:162). The extracellular portion is useful on its own. This can beconfirmed by expression in mammalian cells and sequencing of the cleavedproduct. The signal peptide region was predicted using the NeuralNetwork SignalP V 1.1 program (Nielsen et al, Int. J. Neural Syst.8:581-599 (1997)). One of skill in the art will recognize that theactual cleavage site may be different than that predicted by thecomputer program. SEQ ID NO: 163 is the resulting peptide when thesignal peptide is removed from SEQ ID NO: 160.

The fifth adiponectin-like polypeptide of SEQ ID NO: 186 is anapproximately 288-amino acid protein with a predicted molecular mass ofapproximately 32-kDa unglycosylated. The initial methionine starts atposition 18 of SEQ ID NO: 185 and the putative stop codon begins atpositions 882 of SEQ ID NO: 185. Protein database searches with theBLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altschul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 186 is homologous toadiponectin. Using the Pfam software program (Sonnhammer et al., NucleicAcids Res., 26:320-322 (1998) herein incorporated by reference),adiponectin-like polypeptide of SEQ ID NO: 186 revealed its structuralhomology to C1q and collagen domains. Further description of the Pfammodels can be found at http://pfam.wustl.edu/.

FIG. 9 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 186 and adiponectin SEQ ID NO:402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the twosequences share 63% similarity over 204 amino acid residues and 50%identity over the same 204 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

FIG. 10 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 186 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1), indicating that the two sequencesshare 63% similarity over 204 amino acid residues and 50% identity overthe same 204 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), adiponectin-like polypeptide of SEQ ID NO: 186 wasdetermined to have following eMATRIX domain hits. The results describe:corresponding SEQ ID NO: in sequence listing, e-value, subtype,Accession number, name, position of the domain in the full-lengthprotein, and the amino acid sequence and are shown in Table 6 belowA=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.TABLE 6 SEQ Amino acid ID Accession sequence (start NO: e−value SubtypeNo. Name and end position) 190 2.750e-26 18.26 BL01113B C1q domainPIKFDKILYNEFNHYD proteins TAAGKFTCHIAGVYY FTYHI (175-211) 191 2.000e−1615.60 PR00007C Complement DQASGGIVLQLKLGD C1q domain EVWLQVT (240-262)signature 192 6.143e−16 13.18 BL01113C C1q domain DQASGGIVLQLKLGDproteins EVWLQ (240-260) 193 1.771e−15 14.16 PR00007B ComplementFTCHIAGVYYFTYHIT C1q domain VFSR (196-216) signature 194 4.064e−13 19.33PR00007A Complement TGPQDMPIKFDKILYN C1q domain EFNHYDTAAGK signature(169-196) 195 5.622e−13 17.99 BL01113A C1q domain GIPGNPGHNGLPGRDproteins GRDGAKGDKGDA (29-56) 196 3.077e−12 17.99 BL01113A C1q domainGLPGPMGPIGKPGPK proteins GEAGPTGPQDMP (149-176) 197 3.455e−11 20.42BL00420A Speract receptor GRDGAKGDKGDAGE repeat proteins PGRPGSPGKDGTSGEdomain proteins (44-73) 198 3.618e−11 20.42 BL00420A Speract receptorGIPGNPGHNGLPGRD repeat proteins GRDGAKGDKGDAGE domain proteins (29-58)199 9.673e−11 20.42 BL00420A Speract receptor GDQGSRGSPGKHGPK repeatproteins GLAGPMGEKGLRGE domain proteins (89-118) 200 1.191e−10 17.99BL01113A C1q domain GHPGIPGNPGHNGLP proteins GRDGRDGAKGDK (26-53) 2011.383e−10 17.99 BL01113A C1q domain GLPGRDGRDGAKGD proteinsKGDAGEPGRPGSP (38-65) 202 3.489e−10 17.99 BL01113A C1q domainGNPGHNGLPGRDGRD proteins GAKGDKGDAGEP (32-59) 203 4.246e−10 20.42BL00420A Speract receptor GHPGIPGNPGHNGLP repeat proteins GRDGRDGAKGDKGDdomain proteins (26-55) 204 7.319e−10 17.99 BL01113A C1q domainGDKGDAGEPGRPGSP proteins GKDGTSGEKGER (50-77) 205 7.934e−10 20.42BL00420A Speract receptor GAKGDKGDAGEPGRP repeat proteins GSPGKDGTSGEKGEdomain proteins (47-76) 206 3.908e−09 20.42 BL00420A Speract receptorGDKGDAGEPGRPGSP repeat proteins GKDGTSGEKGERGA domain proteins (50-79)207 4.323e−09 20.42 BL00420A Speract receptor GPEGPRGNIGPLGPTG repeatproteins LPGPMGPIGKPGP domain proteins (134-163) 208 4.349e−09 9.64PR00007D Complement DDTTFTGFLLF C1q domain (275-286) signature 2096.625e−09 7.47 BL01113D C1q domain TTFTGFLLFS proteins (277-287) 2106.885e−09 17.99 BL01113A C1q domain GAKGDKGDAGEPGRP proteinsGSPGKDGTSGEK (47-74) 211 8.096e−09 17.99 BL01113A C1q domainGSPGKDGTSGEKGER proteins GADGKVEAKGIK (62-89) 212 8.788e−09 17.99BL01113A C1q domain CRQGHPGIPGNPGHN proteins GLPGRDGRDGAK (23-50) 2139.585e−09 20.42 BL00420A Speract receptor GPRGNIGPLGPTGLPG repeatproteins PMGPIGKPGPKGE domain proteins (137-166)

The sixth adiponectin-like polypeptide of SEQ ID NO: 215 is anapproximately 300-amino acid protein with a predicted molecular mass ofapproximately 34-kDa unglycosylated. The initial methionine starts atposition 18 of SEQ ID NO: 214 and the putative stop codon begins atpositions 918 of SEQ ID NO: 214. Protein database searches with theBLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altshul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 215 is homologous toadiponectin. Using the Pfam software program (Sonnhammer et al., NucleicAcids Res., 26:320-322 (1998) herein incorporated by reference),adiponectin-like polypeptide of SEQ ID NO: 215 revealed its structuralhomology to C1q and collagen domains. Further description of the Pfammodels can be found at http://pfam.wustl.edu/.

FIG. 11 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 215 and adiponectin SEQ ID NO:402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the twosequences share 48% similarity over 178 amino acid residues and 32%identity over the same 178 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

FIG. 12 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 215 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1), indicating that the two sequencesshare 50% similarity over 182 amino acid residues and 32% identity overthe same 182 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), adiponectin-like polypeptide of SEQ ID NO: 215 wasdetermined to have following eMATRIX domain hits. The results describe:corresponding SEQ ID NO: in sequence listing, e-value, subtype,Accession number, name, position of the domain in the full-lengthprotein, and the amino acid sequence (both amino- and carboxy-flankingregions have been provided for the ease of viewing) and are shown inTable 7 below wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.TABLE 7 SEQ Amino acid ID Accession sequence (start NO: e−value SubtypeNo. Name and end position) 218 8.909e−14 17.99 BL01113A C1q domainGLPGPMGPIGKPGPK proteins GEAGPTGPQGEP (149-176) 219 5.622e−13 17.99BL01113A C1q domain GIPGNPGHNGLPGRD proteins GRDGAKGDKGDA (29-56) 2203.455e−11 20.42 BL00420A Speract receptor GRDGAKGDKGDAGE repeat proteinsPGRPGSPGKDGTSGE domain proteins (44-73) 221 3.618e−11 20.42 BL00420ASperact receptor GIPGNPGHNGLPGRD repeat proteins GRDGAKGDKGDAGE domainproteins (29-58) 222 9.673e−11 20.42 BL00420A Speract receptorGDQGSRGSPGKHGPK repeat proteins GLAGPMGEKGLRGE domain proteins (89-118)223 1.191e−10 17.99 BL01113A C1q domain GHPGIPGNPGHNGLP proteinsGRDGRDGAKGDK (26-53) 224 1.383e−10 17.99 BL01113A C1q domainGLPGRDGRDGAKGD proteins KGDAGEPGRPGSP (38-65) 225 3.489e−10 17.99BL01113A C1q domain GNPGHNGLPGRDGRD proteins GAKGDKGDAGEP (32-59) 2264.246e−10 20.42 BL00420A Speract receptor GHPGIPGNPGHNGLP repeatproteins GRDGRDGAKGDKGD domain proteins (26-55) 227 7.128e−10 17.99BL01113A C1q domain GKPGPKGEAGPTGPQ proteins GEPGVRGIRGWK (158-185) 2287.319e−10 17.99 BL01113A C1q domain GDKGDAGEPGRPGSP proteinsGKDGTSGEKGER (50-77) 229 7.934e−10 20.42 BL00420A Speract receptorGAKGDKGDAGEPGRP repeat proteins GSPGKDGTSGEKGE domain proteins (47-76)230 2.108e−09 20.42 BL00420A Speract receptor GPKGEAGPTGPQGEP repeatproteins GVRGIRGWKGDRGE domain proteins (161-190) 231 2.108e−09 13.18BL01113C C1q domain DASGSIVLQLKLGDE proteins. MWCV (258-278) 2323.596e−09 17.99 BL01113A C1q domain GPIGKPGPKGEAGPTG proteinsPQGEPGVRGIR (155-182) 233 3.631e−09 15.60 PR00007C ComplementDQASGSIVLQLKLGD C1q domain EMWCVIH (258-280) signature 234 3.908e−0920.42 BL00420A Speract receptor GDKGDAGEPGRPGSP repeat proteinsGKDGTSGEKGERGA domain proteins (50-79) 235 4.323e−09 20.42 BL00420ASperact receptor GPEGPRGNIGPLGPTG repeat proteins LPGPMGPIGKPGP domainproteins (134-163) 236 6.885e−09 17.99 BL01113A C1q domainGAKGDKGDAGEPGRP proteins GSPGKDGTSGEK (47-74) 237 8.096e−09 17.99BL01113A C1q domain GSPGKDGTSGEKGER proteins GADGKVEAKGIK (62-89) 2388.788e−09 17.99 BL01113A C1q domain CRQGHPGIPGNPGHN proteinsGLPGRDGRDGAK (23-50) 239 9.585e−09 20.42 BL00420A Speract receptorGPRGNIGPLGPTGLPG repeat proteins PMGPIGKPGPKGE domain proteins (137-166)

The seventh adiponectin-like polypeptide of SEQ ID NO: 241 is anapproximately 314-amino acid protein with a predicted molecular mass ofapproximately 35-kDa unglycosylated. The initial methionine starts atposition 25 of SEQ ID NO: 240 and the putative stop codon begins atpositions 1024 of SEQ ID NO: 240. Protein database searches with theBLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altschul S. F. et al., J Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 241 is homologous toadiponectin. Using the Pfam software program (Sonnhammer et al., NucleicAcids Res., 26:320-322 (1998) herein incorporated by reference),adiponectin-like polypeptide of SEQ ID NO: 241 revealed its structuralhomology to C1q and collagen domains. Further description of the Pfammodels can be found at http://pfam.wustl.edu/.

FIG. 13 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 241 and adiponectin SEQ ID NO:402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the twosequences share 63% similarity over 202 amino acid residues and 50%identity over the same 202 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

FIG. 14 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 241 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1), indicating that the two sequencesshare 63% similarity over 202 amino acid residues and 49% identity overthe same 202 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), adiponectin-like polypeptide of SEQ ID NO: 241 wasdetermined to have following eMATRIXdomain hits. The results describe:corresponding SEQ ID NO: in sequence listing, e-value, subtype,Accession number, name, position of the domain in the full-lengthprotein, and the amino acid sequence and are shown in Table 8 belowwherein A=Alanine, C=Cysteine, D=Aspartic E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, ne, L=Leucine,M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, nine, S=Serine,T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine. TABLE 8 SEQ IDAccession Amino acid sequence NO: e-value Subtype No. Name (position)244 2.750e−26 18.26 BL01113B C1q domain PIKFDKILYNEFNHYD proteinsTAAGKFTCHIAGVYY FTYHI (220-256) 245 2.000e−16 15.60 PR00007C ComplementDQASGGIVLQLKLGD C1q domain EVWLQVT signature (285-307) 246 6.143e−1613.18 BL01113C C1q domain DQASGGIVLQLKLGD proteins EVWLQ (285-305) 2471.771e−15 14.16 PR00007B Complement FTCHIAGVYYFTYHIT C1q domain VFSR(241-261) signature 248 9.143e−15 19.33 PR00007A ComplementFPSSDRPIKFDKILYNE C1q domain FNHYDTAAGK signature (214-241) 2498.909e−14 17.99 BL01113A C1q domain GLPGPMGPIGKPGPK proteinsGEAGPTGPQGEP (149-176) 250 5.622e−13 17.99 BL01113A C1q domainGIPGNPGHNGLPGRD proteins. GRDGAKGDKGDA (29-56) 251 3.455e−11 20.42BL00420A Speract receptor GRDGAKGDKGDAGE repeat proteins PGRPGSPGKDGTSGEdomain proteins (44-73) 252 3.618e−11 20.42 BL00420A Speract receptorGIPGNPGHNGLPGRD repeat proteins GRDGAKGDKGDAGE domain proteins (29-58)253 9.673e−11 20.42 BL00420A Speract receptor GDQGSRGSPGKHGPK repeatproteins GLAGPMGEKGLRGE domain proteins (89-118) 254 1.191e−10 17.99BL01113A C1q domain GHPGIPGNPGHNGLP proteins GRDGRDGAKGDK (26-53) 2551.383e−10 17.99 BL01113A C1q domain GLPGRDGRDGAKGD proteinsKGDAGEPGRPGSP (38-65) 256 1.957e−10 17.99 BL01113A C1q domainGKPGPKGEAGPTGPQ proteins GEPGVQGIRGWK (158-185) 257 3.489e−10 17.99BL01113A C1q domain GNPGHNGLPGRDGRD proteins GAKGDKGDAGEP (32-59) 2584.246e−10 20.42 BL00420A Speract receptor GHPGIPGNPGHNGLP repeatproteins GRDGRDGAKGDKGD domain proteins (26-55) 259 7.319e−10 17.99BL01113A C1q domain GDKGDAGEPGRPGSP proteins GKDGTSGEKGER (50-77) 2607.934e−10 20.42 BL00420A Speract receptor GAKGDKGDAGEPGRP repeatproteins GSPGKDGTSGEKGE domain proteins (47-76) 261 9.852e−10 20.42BL00420A Speract receptor GPKGEAGPTGPQGEP repeat proteins GVQGIRGWKGDRGEdomain proteins (161-190) 262 3.908e−09 20.42 BL00420A Speract receptorGDKGDAGEPGRPGSP repeat proteins GKDGTSGEKGERGA domain proteins (50-79)263 4.323e−09 20.42 BL00420A Speract receptor GPEGPRGNIGPLGPTG repeatproteins LPGPMGPIGKPGP domain proteins (134-163) 264 4.349e−09 9.64PR00007D Complement DDTTFTGFLLF C1q domain (320-331) signature 2654.462e−09 17.99 BL01113A C1q domain GPIGKPGPKGEAGPTG proteinsPQGEPGVQGIR (155-182) 266 6.625e−09 7.47 BL01113D C1q domain TTFTGFLLFSproteins (322-332) 267 6.885e−09 17.99 BL01113A C1q domainGAKGDKGDAGEPGRP proteins GSPGKDGTSGEK (47-74) 268 8.096e−09 17.99BL01113A C1q domain GSPGKDGTSGEKGER proteins GADGKVEAKGIK (62-89) 2698.788e−09 17.99 BL01113A C1q domain CRQGHPGIPGNPGHN proteinsGLPGRDGRDGAK (23-50) 270 9.585e−09 20.42 BL00420A Speract receptorGPRGNIGPLGPTGLPG repeat proteins PMGPIGKPGPKGE domain proteins (137-166)

The eighth adiponectin-like polypeptide of SEQ ID NO: 272 is anapproximately 306-amino acid protein with a predicted molecular mass ofapproximately 34-kDa unglycosylated. The initial methionine starts atposition 25 of SEQ ID NO: 271 and the putative stop codon begins atpositions 943 of SEQ ID NO: 271. Protein database searches with theBLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altschul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 272 is homologous toadiponectin. Using the Pfam software program (Sonnhammer et al., NucleicAcids Res., 26:320-322 (1998) herein incorporated by reference),adiponectin-like polypeptide of SEQ ID NO: 272 revealed its structuralhomology to C1q and collagen domains. Further description of the Pfammodels can be found at http://pfam.wustl.edu/.

FIG. 15 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 272 and adipose tissue-specificprotein AdipoQ SEQ ID NO: 403 (Sato et al, J. Biol. Chem.276:28849-28856 (2001)), indicating that the two sequences share 71%similarity over 78 amino acid residues and 52% identity over the same 78amino acid residues, wherein A=Alanine, C=Cysteine, D=Aspartic Acid,E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine,K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

FIG. 16 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 272 and adipose tissue-specificprotein AdipoQ SEQ ID NO: 403 (Sato et al, J. Biol. Chem.276:28849-28856 (2001)), indicating that the two sequences share 56%similarity over 100 amino acid residues and 43% identity over the same100 amino acid residues, wherein A=Alanine, C=Cysteine, D=Aspartic Acid,E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine,K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

FIG. 17 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 272 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1), indicating that the two sequencesshare 54% similarity over 200 amino acid residues and 42% identity overthe same 200 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), adiponectin-like polypeptide of SEQ ID NO: 272 wasdetermined to have following eMATRIX domain hits. The results describe:corresponding SEQ ID NO: in sequence listing, e-value, subtype,Accession number, name, position of the domain in the full-lengthprotein, and the amino acid sequence and are shown in Table 9 belowwherein A=Alanine, C=Cysteine, D=Aspartic Acid E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.TABLE 9 SEQ ID Accession Amino acid sequence NO: e-value Subtype No.Name (start and end position) 275 2.000e−16 15.60 PR00007C ComplementDQASGGIVLQLKLGD C1q domain EVWLQVT (258-280) signature 276 6.143e−1613.18 BL01113C C1q domam DQASGGIVLQLKLGD proteins EVWLQ (258-278) 2778.909e−14 17.99 BL01113A C1q domain GLPGPMGPIGKPGPK proteinsGEAGPTGPQGEP (149-176) 278 5.622e−13 17.99 BL01113A C1q domainGIPGNPGHNGLPGRD proteins GRDGAKGDKGDA (29-56) 279 3.455e−11 20.42BL00420A Speract receptor GRDGAKGDKGDAGE repeat proteins PGRPGSPGKDGTSGEdomain proteins (44-73) 280 3.618e−11 20.42 BL00420A Speract receptorGIPGNPGHNGLPGRD repeat proteins GRDGAKGDKGDAGE domain proteins (29-58)281 9.673e−11 20.42 BL00420A Speract receptor GDQGSRGSPGKHGPK repeatproteins GLAGPMGEKGLRGE domain proteins (89-118) 282 1.191e−10 17.99BL01113A C1q domain GHPGIPGNPGHNGLP proteins GRDGRDGAKGDK (26-53) 2831.383e−10 17.99 BL01113A C1q domain GLPGRDGRDGAKGD proteinsKGDAGEPGRPGSP (38-65) 284 1.957e−10 17.99 BL01113A C1q domainGKPGPKGEAGPTGPQ proteins GEPGVQGIRGWK (158-185) 285 3.489e−10 17.99BL01113A C1q domain GNPGHNGLPGRDGRD proteins GAKGDKGDAGEP (32-59) 2864.246e−10 20.42 BL00420A Speract receptor GHPGIPGNPGHNGLP repeatproteins GRDGRDGAKGDKGD domain proteins (26-55) 287 7.319e−10 17.99BL01113A C1q domain GDKGDAGEPGRPGSP proteins GKDGTSGEKGER (50-77) 2887.934e−10 20.42 BL00420A Speract receptor GAKGDKGDAGEPGRP repeatproteins GSPGKDGTSGEKGE domain proteins (47-76) 289 9.852e−10 20.42BL00420A Speract receptor GPKGEAGPTGPQGEP repeat proteins GVQGIRGWKGDRGEdomain proteins (161-190) 290 3.908e−09 20.42 BL00420A Speract receptorGDKGDAGEPGRPGSP repeat proteins GKDGTSGEKGERGA domain proteins (50-79)291 4.323e−09 20.42 BL00420A Speract receptor GPEGPRGNIGPLGPTG repeatproteins LPGPMGPIGKPGP domain proteins (134-163) 292 4.349e−09 9.64PR00007D Complement DDTTFTGFLLF C1q domain (293-304) signature 2934.462e−09 17.99 BL01113A C1q domain GPIGKPGPKGEAGPTG proteinsPQGEPGVQGIR (155-182) 294 6.625e−09 7.47 BL01113D C1q domain TTFTGFLLFS(295-305) proteins 295 6.885e−09 17.99 BL01113A C1q domainGAKGDKGDAGEPGRP proteins GSPGKDGTSGEK (47-74) 296 8.096e−09 17.99BL01113A C1q domain GSPGKDGTSGEKGER proteins GADGKVEAKGIK (62-89) 2978.788e−09 17.99 BL01113A C1q domain CRQGHPGIPGNPGHN proteinsGLPGRDGRDGAK (23-50) 298 9.585e−09 20.42 BL00420A Speract receptorGPRGNIGPLGPTGLPG repeat proteins PMGPIGKPGPKGE domain proteins (137-166)

A predicted approximately nineteen-residue signal peptide is encodedfrom approximately residue 1 to residue 19 of SEQ ID NO: 186, 215, 241,and 272 (SEQ ID NO: 188). The extracellular portion is useful on itsown. This can be confirmed by expression in mammalian cells andsequencing of the cleaved product. The signal peptide region waspredicted using the Neural Network SignalP V1.1 program (Nielsen et al,Int. J. Neural Syst. 8:581:599 (1997)). One of skill in the art willrecognize that the actual cleavage site may be different than thatpredicted by the computer program. SEQ ID NO: 189 is the resultingpeptide when the signal peptide is removed from SEQ ID NO: 186. SEQ IDNO: 217 is the resulting peptide when the signal peptide is removed fromSEQ ID NO: 215. SEQ ID NO: 243 is the resulting peptide when the signalpeptide is removed from SEQ ID NO: 241. SEQ ID NO: 274 is the resultingpeptide when the signal peptide is removed from SEQ ID NO: 272.

The ninth adiponectin-like polypeptide of SEQ ID NO: 302 is anapproximately 338-amino acid protein with a predicted molecular mass ofapproximately 38-kDa unglycosylated. The initial methionine starts atposition 199 of SEQ ID NO: 301 and the putative stop codon begins atpositions 1213 of SEQ ID NO: 301. Protein database searches with theBLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altschul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 301 is homologous toadiponectin. Using the Pfam software program (Sonnhammer et al., NucleicAcids Res., 26:320-322 (1998) herein incorporated by reference),adiponectin-like polypeptide of SEQ ID NO: 302 revealed its structuralhomology to C1q and collagen domains. Further description of the Pfammodels can be found at http://pfam.wustl.edu/.

FIG. 18 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 302 and adiponectin SEQ ID NO:402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the twosequences share 52% similarity over 220 amino acid residues and 37%identity over the same 220 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

FIG. 19 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 302 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1), indicating that the two sequencesshare 53% similarity over 220 amino acid residues and 37% identity overthe same 220 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), adiponectin-like polypeptide of SEQ ID NO: 302 wasdetermined to have following eMATRIX domain hits. The results describe:corresponding SEQ ID NO: in sequence listing, e-value, subtype,Accession number, name, position of the domain in the full-lengthprotein, and the amino acid sequence and shown in Table 10 below whereinA=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.TABLE 10 SEQ ID Accession Amino acid sequence NO: e-value Subtype No.Name (start and end position) 304 3.647e−27 18.26 BL01113B C1q domainVLKFDDVVTNLGNHY proteins DPTTGKFTCSIPGIYFF TYHV (225-261) 305 6.657e−1514.16 PR00007B Complement FTCSIPGIYFFTYHVL C1q domain MRGG (246-266)signature 306 2.047e−14 15.60 PR00007C Complement DYASNSVVLHLEPGD C1qdomain EVYIKLD (294-316) signature 307 1.000e−13 17.99 BL01113A C1qdomain GEPGPPGPMGPPGEK proteins GEPGRQGLPGPP (162-189) 308 2.532e−1313.18 BL01113C C1q domain DYASNSVVLHLEPGD proteins EVYIK (294-314) 3097.081e−13 17.99 BL01113A C1q domain GKAGPRGPPGEPGPP proteinsGPMGPPGEKGEP (153-180) 310 8.297e−13 17.99 BL01113A C1q domainGRPGKAGPRGPPGEP proteins GPPGPMGPPGEK (150-177) 311 3.538e−12 17.99BL01113A C1q domain GPPGEPGPPGPMGPPG proteins EKGEPGRQGLP (159-186) 3124.808e−12 20.42 BL00420A Speract receptor GRPGKAGPRGPPGEP repeatproteins GPPGPMGPPGEKGE domain proteins (150-179) 313 5.385e−12 17.99BL01113A C1q domain GPPGPMGPPGEKGEP proteins GRQGLPGPPGAP (165-192) 3148.412e−12 19.33 PR00007A Complement QHEGYEVLKFDDVVT C1q domainNLGNHYDPTTGK signature (219-246) 315 5.909e−11 17.99 BL01113A C1q domainGPMGPPGEKGEPGRQ proteins GLPGPPGAPGLN (168-195) 316 8.773e−11 17.99BL01113A C1q domain GPRGPPGEPGPPGPM proteins GPPGEKGEPGRQ (156-183) 3178.967e−10 20.42 BL00420A Speract receptor GEAGRPGKAGPRGPP repeatproteins GEPGPPGPMGPPGE domain proteins (147-176) 318 7.231e−09 20.42BL00420A Speract receptor GPPGPMGPPGEKGEP repeat proteins GRQGLPGPPGAPGLdomain proteins (165-194) 319 7.307e−09 4.29 BL00415N SynapsinsPRGPPGEPGPPGPMGP proteins PGEKGEPGRQGLPGPP GAPGLNAAGAIS (157-201) 3209.135e-09 17.99 BL01113A C1q domain GEAGRPGKAGPRGPP proteinsGEPGPPGPMGPP (147-174) 321 9.169e-09 20.42 BL00420A Speract receptorGPPGEKGEPGRQGLP repeat proteins GPPGAPGLNAAGAI domain proteins (171-200)

The tenth adiponectin-like polypeptide of SEQ ID NO: 323 is anapproximately 244-amino acid protein with a predicted molecular mass ofapproximately 27-kDa unglycosylated. The initial methionine starts atposition 161 of SEQ ID NO: 322 and the putative stop codon begins atpositions 893 of SEQ ID NO: 322. Protein database searches with theBLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altschul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 323 is homologous toadiponectin. Using the Pfam software program (Sonnhammer et al., NucleicAcids Res., 26:320-322 (1998) herein incorporated by reference),adiponectin-like polypeptide of SEQ ID NO: 323 revealed its structuralhomology to C1q and collagen domains. Further description of the Pfammodels can be found at http://pfam.wustl.edu/.

FIG. 20 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 323 and adiponectin SEQ ID NO:402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the twosequences share 52% similarity over 220 amino acid residues and 37%identity over the same 220 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

FIG. 21 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 323 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1), indicating that the two sequencesshare 53% similarity over 220 amino acid residues and 37% identity overthe same 220 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), adiponectin-like polypeptide of SEQ ID NO: 323 wasdetermined to have following eMATRIX domain hits. The results describe:corresponding SEQ ID NO: in sequence listing, e-value, subtype,Accession number, name, position of the domain in the full-lengthprotein, and the amino acid sequence and are shown in Table 11 belowwherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.TABLE 11 SEQ ID Accession Amino acid sequence NO: e-value Subtype No.Name (start and end position) 327 3.647e−27 18.26 BL01113B C1q domainVLKFDDVVTNLGNHY proteins DPTTGKFTCSIPGIYFF TYHV (131-167) 328 6.657e−1514.16 PR00007B Complement FTCSIPGIYFFTYHVL C1q domain MRGG (152-172)signature 329 2.047e−14 15.60 PR00007C Complement DYASNSVVLHLEPGD C1qdomain EVYIKLD (200-222) signature 330 1.000e−13 17.99 BL01113A C1qdomain GEPGPPGPMGPPGEK proteins GEPGRQGLPGPP (68-95) 331 2.532e−13 13.18BL01113C C1q domain DYASNSVVLHLEPGD proteins EVYIK (200-220) 3327.081e−13 17.99 BL01113A C1q domain GKAGPRGPPGEPGPP proteinsGPMGPPGEKGEP (59-86) 333 8.297e−13 17.99 BL01113A C1q domainGRPGKAGPRGPPGEP proteins GPPGPMGPPGEK (56-83) 334 3.538e−12 17.99BL01113A C1q domain GPPGEPGPPGPMGPPG proteins EKGEPGRQGL (65-92) 3354.808e−12 20.42 BL00420A Speract receptor GRPGKAGPRGPPGEP repeatproteins GPPGPMGPPGEKGE domain proteins (56-85) 336 5.385e−12 17.99BL01113A C1q domain GPPGPMGPPGEKGEP proteins GRQGLPGPPGAP (71-98) 3378.412e−12 19.33 PR00007A Complement QHEGYEVLKFDDVVT C1q domainNLGNHYDPTTGK signature (125-152) 338 5.909e−11 17.99 BL01113A C1q domainGPMGPPGEKGEPGRQ proteins GLPGPPGAPGLN (74-101) 339 8.773e−11 17.99BL01113A C1q domain GPRGPPGEPGPPGPM proteins GPPGEKGEPGRQ (62-89) 3408.967e−10 20.42 BL00420A Speract receptor GEAGRPGKAGPRGPP repeatproteins GEPGPPGPMGPPGE domain proteins (53-82) 341 7.231e−09 20.42BL00420A Speract receptor GPPGPMGPPGEKGEP repeat proteins GRQGLPGPPGAPGLdomain proteins (71-100) 342 7.307e−09 4.29 BL00415N SynapsinsPRGPPGEPGPPGPMGP proteins PGEKGEPGRQGLPGPP GAPGLNAAGAIS (63-107) 3439.135e−09 17.99 BL01113A C1q domain GEAGRPGKAGPRGPP proteinsGEPGPPGPMGPP (53-80) 344 9.169e−09 20.42 BL00420A Speract receptorGPPGEKGEPGRQGLP repeat proteins GPPGAPGLNAAGAI domain proteins (77-106)

A predicted approximately nineteen-residue signal peptide is encodedfrom approximately residue 1 to residue 19 of SEQ ID NO: 323 (SEQ ID NO:325). The extracellular portion is useful on its own. This can beconfirmed by expression in mammalian cells and sequencing of the cleavedproduct. The signal peptide region was predicted using the NeuralNetwork SignalP V1.1 program (Nielsen et al, Int. J. Neural Syst.8:581-599 (1997)). One of skill in the art will recognize that theactual cleavage site may be different than that predicted by thecomputer program. SEQ ID NO: 326 is the resulting peptide when thesignal peptide is removed from SEQ ID NO: 323.

The eleventh adiponectin-like polypeptide of SEQ ID NO: 348 is anapproximately 513-amino acid protein with a predicted molecular mass ofapproximately 57-kDa unglycosylated. The initial methionine starts atposition 1 of SEQ ID NO: 347 and the putative stop codon begins atpositions 1540 of SEQ ID NO: 347. Protein database searches with theBLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altschul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 348 is homologous toadiponectin. Using the Pfam software program (Sonnhammer et al., NucleicAcids Res., 26:320-322 (1998) herein incorporated by reference),adiponectin-like polypeptide of SEQ ID NO: 348 revealed its structuralhomology to C1q and collagen domains. Further description of the Pfammodels can be found at http://pfam.wustl.edu/.

FIG. 22 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 348 and adiponectin SEQ ID NO:402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the twosequences share 40% similarity over 220 amino acid residues and 31%identity over the same 220 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

FIG. 23 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 348 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1), indicating that the two sequencesshare 40% similarity over 243 amino acid residues and 30% identity overthe same 243 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), adiponectin-like polypeptide of SEQ ID NO: 348 wasdetermined to have following eMATRIX domain hits. The results describe:corresponding SEQ ID NO: in sequence listing, e-value, subtype,Accession number, name, position of the domain in the full-lengthprotein, and the amino acid sequence and are shown in Table 12 belowwherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.TABLE 12 SEQ ID Accession Amino acid sequence NO: e-value Subtype No.Name (start and end position) 350 5.421e−16 18.26 BL01113B C1q domainVVLFNKVLVNDGDVYNP proteins STGVFTAPYDGRYLITAT L (383-419) 351 8.568e−1419.33 PR00007A Complement FPSDGGVVLFNKVLVND C1q domain GDVYNPSTGV(377-404) signature

The twelfth adiponectin-like polypeptide of SEQ ID NO: 355 is anapproximately 293-amino acid protein with a predicted molecular mass ofapproximately 33-kDa unglycosylated. The initial methionine starts atposition 683 of SEQ ID NO: 354 and the putative stop codon begins atpositions 1556 of SEQ ID NO: 354. Protein database searches with theBLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altschul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 355 is homologous toadiponectin. Using the Pfam software program (Sonnhammer et al., NucleicAcids Res., 26:320-322 (1998) herein incorporated by reference),adiponectin-like polypeptide of SEQ ID NO: 355 revealed its structuralhomology to C1q and collagen domains. Further description of the Pfammodels can be found at http://pfam.wustl.edu/.

FIG. 24 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 355 and adiponectin SEQ ID NO:402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the twosequences share 50% similarity over 134 amino acid residues and 39%identity over the same 134 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

FIG. 25 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 355 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1), indicating that the two sequencesshare 51% similarity over 134 amino acid residues and 40% identity overthe same 134 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), adiponectin-like polypeptide of SEQ ID NO: 355 wasdetermined to have following eMATRIX domain hits. The results describe:corresponding SEQ ID NO: in sequence listing, e-value, subtype,Accession number, name, position of the domain in the full-lengthprotein, and the amino acid sequence and are shown in Table 13 belowwherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.TABLE 13 SEQ ID Accession Amino acid sequence NO: e-value Subtype No.Name (start and end position) 359 3.786e−23 18.26 BL01113B C1q domainVLRFDDVVTNVGNA proteins YEAASGKFTCPMPGV YFFAYHV (125-161) 360 5.114e−1514.16 PR00007B Complement FTCPMPGVYFFAYHV C1q domain LMRGG (146-166)signature 361 7.968e−15 17.99 BL01113A C1q domain GPPGPRGPPGEPGRPGproteins PPGPPGPGPGG (73-100) 362 5.091e−14 17.99 BL01113A C1q domainGPPGPPGPRGPPGEPG proteins RPGPPGPPGPG (70-97) 363 5.295e−11 17.99BL01113A C1q domain GKAGLRGPPGPPGPR proteins GPPGEPGRPGPP (64-91) 3648.568e−11 17.99 BL01113A C1q domain GPPGEPGRPGPPGPPG proteinsPGPGGVAPAAG (79-106) 365 8.691e−11 20.42 BL00420A Speract receptorGPPGPRGPPGEPGRPG repeat proteins PPGPPGPGPGGVA domain proteins (73-102)366 8.977e−11 17.99 BL01113A C1q domain GLRGPPGPPGPRGPPG proteinsEPGRLPGPPGPP (67-94) 367 9.673e−11 20.42 BL00420A Speract receptorGPPGPPGPRGPPGEPG repeat proteins RPGPPGPPGPGPG domain proteins (70-99)368 2.180e−10 20.42 BL00420A Speract receptor GAKGEVGRRGKAGL repeatproteins RGPPGPPGPRGPPGE domain proteins (55-84) 369 7.052e−10 19.33PR00007A Complement PHEGYEVLRFDDVVT C1q domain NVGNAYEAASGK Signature(119-146) 370 4.351e−09 5.36 PR00524F Cholecystokinin GPPGPPGPRGPPGEtype A receptor (70-84) signature 371 4.635e−09 17.99 BL01113A C1qdomain GEPGRPGPPGPPGPGP proteins GGVAPAAGYVP (82-109) 372 6.192e−0917.99 BL01113A C1q domain GPRGPPGEPGRPGPPG proteins PPGPGPGGVAP (76-103)373 6.595e−09 13.84 DM00250B Kw Annexin GEPGRPGPPGPPGPGP antiben prolineGGVAPAAG (82-106) tumor 374 7.372e−09 4.29 BL00415N SynapsinsRRGKAGLRGPPGPPG proteins PRGPPGEPGRPGPPGP PGPGPGGVAPAAG (62-106) 3757.750e−09 17.99 BL01113A C1q domain GRRGKAGLRGPPGPP proteinsGPRGPPGEPGRPGPP (61-88) 376 8.062e−09 20.42 BL00420A Speract receptorFPPGAKGEVGRRGKA repeat proteins GLRGPPGPPGPRGP domain proteins (52-81)

The thirteenth adiponectin-like polypeptide of SEQ ID NO: 378 is anapproximately 238-amino acid protein with a predicted molecular mass ofapproximately 27-kDa unglycosylated. The initial methionine starts atposition 683 of SEQ ID NO: 377 and the putative stop codon begins atpositions 1391 of SEQ ID NO: 377. Protein database searches with theBLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altschul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 378 is homologous toadiponectin. Using the Pfam software program (Sonnhammer et al., NucleicAcids Res., 26:320-322 (1998) herein incorporated by reference),adiponectin-like polypeptide of SEQ ID NO: 355 revealed its structuralhomology to C1q and collagen domains. Further description of the Pfammodels can be found at http://pfam.wustl.edu/.

FIG. 26 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 378 and adiponectin SEQ ID NO:402 (Hotta et al, Diabetes 50:1126-1133 (2001)), indicating that the twosequences share 52% similarity over 215 amino acid residues and 37%identity over the same 215 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

FIG. 27 shows the BLASTP amino acid sequence alignment betweenadiponectin-like polypeptide SEQ ID NO: 378 and human adiponectin SEQ IDNO: 404 (Patent No. JP3018186-B1), indicating that the two sequencesshare 53% similarity over 215 amino acid residues and 38% identity overthe same 215 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), adiponectin-like polypeptide of SEQ ID NO: 378 wasdetermined to have following eMATRIX domain hits. The results describe:corresponding SEQ ID NO: in sequence listing, e-value, subtype,Accession number, name, position of the domain in the full-lengthprotein, and the amino acid sequence and are shown in Table 14 belowwherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.TABLE 14 SEQ ID Accession Amino acid sequence NO: e-value Subtype No.Name (start and end position) 381 3.786e−23 18.26 BL01113B C1q domainVLRFDDVVTNVGNA proteins YEAASGKFTCPMPGV YFFAYHV (125-161) 382 5.114e−1514.16 PR00007B Complement FTCPMPGVYFFAYHV C1q domain LMRGG (146-166)signature 383 7.968e−15 17.99 BL01113A C1q domain GPPGPRGPPGEPGRPGproteins PPGPPGPGPGG (73-100) 384 5.091e−14 17.99 BL01113A C1q domainGPPGPPGPRGPPGEPG proteins RPGPPGPPGPG (70-97) 385 5.875e−13 15.60PR00007C Complement DYASNSVILHLDVGD C1q domain EVFIKLD (194-216)signature 386 4.000e−12 13.18 BL01113C C1q domain DYASNSVILHLDVGDproteins EVFLK (194-214) 387 5.295e−11 17.99 BL01113A C1q domainGKAGLRGPPGPPGPR proteins GPPGEPGRPGPPGPP (64-91) 388 8.568e−11 17.99BL01113A C1q domain GPPGEPGRPGPPGPPG proteins PGPGGVAPAAG (79-106) 3898.691e−11 20.42 BL00420A Speract receptor GPPGPRGPPGEPGRPG repeatproteins PPGPPGPGPGGVA domain proteins (73-102) 390 8.977e−11 17.99BL01113A C1q domain GLRGPPGPPGPRGPPG proteins EPGRPGPPGPP (67-94) 3919.673e−11 20.42 BL00420A Speract receptor GPPGPPGPRGPPGEPG repeatproteins RPGPPGPPGPGPG domain proteins (70-99) 392 2.180e−10 20.42BL00420A Speract receptor GAKGEVGRRGKAGL repeat proteins RGPPGPPGPRGPPGEdomain proteins (55-84) 393 7.052e−10 19.33 PR00007A ComplementPHEGYEVLRFDDVVT C1q domain NVGNAYEAASGK signature (119-146) 3944.351e−09 5.36 PR00524F Cholecystokinin GPPGPPGPRGPPGE type A receptor(70-84) signature 395 4.635e−09 17.99 BL01113A C1q domainGEPGRPGPPGPPGPGP proteins GGVAPAAGYVP (82-109) 396 6.192e−09 17.99BL01113A C1q domain GPRGPPGEPGRPGPPG proteins PPGPGPGGVAP (76-103) 3976.595e−09 13.84 DM00250B kw Annexin GEPGRPGPPGPPGPGP antigen prolineGGVAPAAG (82-106) tumor 398 7.372e−09 4.29 BL00415N SynapsinsRRGKAGLRGPPGPPG proteins PRGPPGEPGRPGPPGP PGPGPGGVAPAAG (62-106) 3997.750e−09 17.99 BL01113A C1q domain GRRGKAGLRGPPGPP proteinsGPRGPPGEPGRP (61-88) 400 7.750e−09 7.47 BL01113D C1q domain STFSGFIIYP(228-238) proteins 401 8.062e−09 20.42 BL00420A Speract receptorFPPGAKGEVGRRGKA repeat proteins GLRGPPGPPGPRGP domain proteins (52-81)

A predicted approximately fifteen-residue signal peptide is encoded fromapproximately residue 1 to residue 15 of SEQ ID NO: 355, or 378 (SEQ IDNO: 357). The extracellular portion is useful on its own. This can beconfirmed by expression in mammalian cells and sequencing of the cleavedproduct. The signal peptide region was predicted using the NeuralNetwork SignalP V1.1 program (Nielsen et al, Int. J. Neural Syst.8:581-599 (1997)). One of skill in the art will recognize that theactual cleavage site may be different than that predicted by thecomputer program. SEQ ID NO: 358 is the resulting peptide when thesignal peptide is removed from SEQ ID NO: 355. SEQ ID NO: 380 is theresulting peptide when the signal peptide is removed from SEQ ID NO:378.

The adiponectin-like polypeptides and polynucleotides of the inventionmay be used to treat obesity, diabetes, lipoatrophy, coronary arterydiseases, atherosclerosis, and other obesity and diabetes-relatedcardiovascular pathologies. Adiponectin-like polypeptides andpolynucleotides of the invention may also be used in treatment ofautoimmune diseases and inflammation, to modulate immune responses, andto treat transplant patients.

4.2 Serpin-Like Polypeptides and Polynucleotides

Proteinases play many important physiological functions in the body,including food digestion, remodeling of extracellular matrices, bloodcoagulation, and immune processes (Salzet et al., Trends Immunol.20:541-544 (1999), herein incorporated by reference in its entirety).Proteinases have also been implicated in maturation of signalingproteins (e.g. methionine enkaphalin), hormones, and digestive enzymes.Proteinases are classified based on the central amino acid residue inthe active site of the proteinase (like serine proteinases, cysteineproteinases, or aspartate proteinases). Proteinases are implicated inmany pathologies including emphysema, arthritis, and cardiovasculardiseases. Proteinases are regulated by binding of inhibitory proteins inthe extracellular environment.

Serpins (serine proteinase inhibitors) are a superfamily of more than500 proteins, about 350-500 amino acids in size, that fold into aconserved structure and employ a unique suicide substrate-likeinhibitory strategy (Silverman et al., J. Biol. Chem. 276:33293-33296(2001), herein incorporated by reference in its entirety). The serpinsuperfamily has evolved over 500 million years with representativesfound in viruses, plants, protozoa, insects, and higher vertebrates(Schich et al., J. Biol. Chem. 272:1849-1855 (1997), herein incorporatedby reference in its entirety). The tertiary structures of serpinsdemonstrate 3β-sheets, ˜9α-helices, and several loops that are arrangedinto a metastable conformation (Askew et al., J. Biol. Chem.276:49320-49330 (2001), herein incorporated by reference in itsentirety). The mobile reactive site loop (RSL) is displayed on thesurface, and serves as pseudo-substrate to bind to proteinase. Uponbinding to proteinase and cleavage of the RSL loop the serpin moleculeundergoes a conformational change that traps the proteinase in acovalent acyl-enzyme intermediate. Serpins regulate serine proteinasesinvolved in coagulation, fibrinolysis, inflammation, cell migration, andextracellular matrix remodeling.

A subclass of serpins exhibits strong sequence similarity to chickenovalbumin. The serpin-like molecule of present invention which hasstrong homology to SERPINB12, belong to this subclass of serpins. Theseov-serpins lack both the N-terminal signal peptides and C-terminalextensions of other serpins. They also exhibit a variable length loopbetween C and D helices that may harbor functional motifs. Theov-serpins are proposed to be either cytoplasmic or nucleocytoplasmicproteins. However, many of them (maspin, megsin, and SCCAs) may functionextracellularly as they are released from cells under certainconditions. The ov-serpins are functional inhibitors of serine orcysteine proteinases. Many of them inhibit more than one class ofproteinases. Many of the ov-serpins are present in the same cells thatsecrete the proteinases and thus may have regulatory functions. They mayalso help protect the secreting cell from the proteinases.

Thus, the Serpin-like polypeptides and polynucleotides of the inventionmay be used to treat emphysema, arthritis, blood clotting disorders, andcardiovascular disease. Serpin-like polypeptides and polynucleotides ofthe invention may also be used in treatment of immune disorders andinflammation, to modulate immune responses, and to treat transplantpatients. Serpin-like polypeptides may also be useful as marker indiagnosis and prognosis of certain cancers.

The Serpin-like polypeptide of SEQ ID NO: 408 is an approximately425-amino acid protein with a predicted molecular mass of approximately48-kDa unglycosylated. The initial methionine starts at position 78 ofSEQ ID NO: 407 and the putative stop codon begins at positions 1353 ofSEQ ID NO: 407. Protein database searches with the BLASTP algorithm(Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al., J. Mol. Biol. 21:403-10 (1990), herein incorporated byreference in their entirety) indicate that SEQ ID NO: 408 is homologousto SERPINB12 and squamous cell carcinoma antigen 2 (SCCA2). Using thePfam software program (Sonnhammer et al., Nucleic Acids Res. 26:320-322(1998), herein incorporated by reference in its entirety), Serpin-likepolypeptide of SEQ ID NO: 408 revealed its sequence homology to serpins.Further description of the Pfam models can be found athttp://pfam.wustl.edu/.

FIG. 28 shows the BLASTP amino acid sequence alignment of the first highscoring pair (HSP) between Serpin-like polypeptide SEQ ID NO: 408 andSERPINB12 SEQ ID NO: 416 (Askew et al., J. Biol. Chem. 276:49320-49330(2001), herein incorporated by reference in its entirety), indicatingthat the two sequences share 99% similarity over 326 amino acid residuesand 99% identity over the same 326 amino acid residues, whereinA=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

FIG. 29 shows the BLASTP amino acid sequence alignment of the secondhigh scoring pair (HSP) between Serpin-like polypeptide SEQ ID NO: 408and SERPINB12 SEQ ID NO: 416 (Askew et al., J. Biol. Chem.276:49320-49330 (2001), herein incorporated by reference in itsentirety), indicating that the two sequences share 100% similarity over81 amino acid residues and 100% identity over the same 81 amino acidresidues, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

FIG. 30 shows the BLASTP amino acid sequence alignment of the first highscoring pair (HSP) between Serpin-like polypeptide SEQ ID NO: 408 andhuman SCCA2 protein SEQ ID NO: 417 (Patent No. DE19742725-A1, hereinincorporated by reference in its entirety), indicating that the twosequences share 65% similarity over 336 amino acid residues and 48%identity over the same 336 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

FIG. 31 shows the BLASTP amino acid sequence alignment of the secondhigh scoring pair (HSP) between Serpin-like polypeptide SEQ ID NO: 408and human SCCA2 protein SEQ ID NO: 417 (Patent No. DE19742725-A1, hereinincorporated by reference in its entirety), indicating that the twosequences share 78% similarity over 70 amino acid residues and 51%identity over the same 70 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference in its entirety), Serpin-like polypeptide of SEQ ID NO: 408was determined to have following eMATRIX domain hits. The resultsdescribe: corresponding SEQ ID NO: in sequence listing, e-value,subtype, Accession number, name, position of the domain in thefull-length protein, and the amino acid sequence and are shown in Table15 below wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.TABLE 15 Amino SEQ Acid Sequence ID sub- Accession (start and NO:e-value type No. Name end position) 410 7.600e−25 28.56 BL00284C SerpinsTVLVLVNAVYFKA proteins KWETYFDHENTVD APFCLNANENKSV KMM (203-245) 4114.375e−23 19.15 BL00284E Serpins NHPFLFFIRHNKT proteins QTILFYGRVCSP(401-426) 412 5.286e−21 16.34 BL00284D Serpins LSFPRFTLEGSYD proteinsLNSILQDMGITDI F (317-344) 413 6.192e−17 15.64 BL00284A SerpinsNIFFSPLSLSAAL proteins GMVRLGARSDS (27-51) 414 4.414e−13 17.99 BL00284BSerpins SRQEINFWVECQS proteins QGKIKELF (174-195)

Serpins undergo a conformational change upon binding of the proteinasesubstrate thereby trapping the proteinase in a covalent acyl-enzymeintermediate (Huntington et al., Nature 407:923-926 (2000), hereinincorporated by reference). Serpins utilize this mechanism to regulateproteinase cascades involved in blood clotting, fibrinolysis, complementactivation, cell motility, inflammation, and cell death (Silverman etal., J. Biol. Chem. 276:33293-33296 (2001); Carrell et al., Mol. Biol.Med. 6:35-42 (1989); Potempa et al., J. Biol. Chem. 269:15957-15960(1994), all of which are herein incorporated by reference). Members ofthe ov-serpin subfamily inhibit various serine or cysteine proteinasesand are involved in inhibition of cell migration, protection againstapoptosis, and neutralization of endogenous granule proteinases thatleak into the cytosol (Silverman et al., J. Biol. Chem. 276:33293-33296(2001); Bird, Immunol. Cell. Biol. 77:47-57 (1999), both of which areherein incorporated by reference). Specifically, SERPINB12 is a potentinhibitor of trypsin-like serine proteinases, including trypsin andplasmin (Askew et al., J. Biol. Chem. 276:49320-49330 (2001), hereinincorporated by reference).

The polypeptides of the invention are expected to have similar functionsas serpins, specifically the ov-serpins such as SERPINB12, acting as aninhibitor of serine and cysteine proteinases. The polypeptides,polynucleotides, antibodies, and other compositions of the invention areexpected to be useful in treating the following disorders: emphysema,arthritis, blood clotting disorders and cardiovascular diseases.Serpin-like polypeptides and polynucleotides of the invention may alsobe used in the treatment of immune disorders and inflammation, tomodulate immune responses, and to treat transplant patients. Serpin-likepolypeptides may also be useful as markers in diagnosis and prognosis ofcertain cancers.

4.3 Nogo-Receptor-Like (NgRHy) Polypeptides and Polynucleotides

The establishment of neural connections during development is a highlydynamic process. A key aspect of this process is the regulation of axongrowth, which is mediated by a variety of chemotropic factors (Skaper,et al., Prog. Neurobiol. 56:593-608 (2001), herein incorporated byreference). Chemotropism, which determines the direction of axonalgrowth, results from the concerted action of chemoattractant andchemorepellent cues (Yu and Bargmann, Nat. Neurosci. 4(Suppl.):1169-1176(2001), herein incorporated by reference). Growth cones, the leadingedge of the axons, encounter and detect these guiding cues along theirtrajectories in the form of gradients of diffusible factors, necessaryfor long-range guidance (Zheng and Kuffler, J. Neurobiol. 42:212-219(2000), herein incorporated by reference), extracellularmatrix-associated molecules, required for both short- and long-rangeregulation (Hynds and Snow, Exp. Neurol. 160:244-255 (1999), hereinincorporated by reference; Skaper et al., supra), and membrane-boundmolecules, necessary for short-range regulation (He and Meini, Mol.Cell. Neurosci. 19:18-31 (2002), herein incorporated by reference). Itis believed that the inability of mature neurons to regenerateappropriate connections following injury or trauma is in part mediatedby chemorepellent molecules present along axonal tracts (Fawcett, CellTissue Res. 290:371-377 (1997), herein incorporated by reference).

Results from studies demonstrating that neurons in the adult centralnervous system (CNS) have regenerative potential support thishypothesis. For example, it is known that severed fibers of the opticnerve and of the spinal cord are unable to regenerate across the site oflesion (reviewed in Tessler-Lavigne and Goodman, Science 287:813-814(2000), herein incorporated by reference). In contrast, injuries do notprevent motor and sensory neurons projections to peripheral targets toregenerate. Experiments by David and Aguayo (Science 214:931-933 (1981),herein incorporated by reference) indicate that if the two extremitiesof severed optic nerves are “bridged” surgically with a graft obtainedfrom peripheral nerves, retinal projection re-growth along the graftedfibers extends well beyond the injured site. These and other experimentsled to the hypothesis that the myelin sheath surrounding CNS axonscontains inhibitory cues that are absent in myelin of axons in theperipheral nervous system (PNS) (Schwab and Caroni, J. Neurosci. 8:2381-2393 (1988), herein incorporated by reference).

The search for inhibitory cues present in CNS myelin preparation, led tothe identification of an inhibitory activity found only in CNS myelin(GrandPré and Strittmatter, Neuroscientist 7:377-386 (2001), hereinincorporated by reference). Protein purification combined withinhibitory activity in vitro assays identified myelin protein fractionsof approximately 35 and 250 kD, known as neurite growth inhibitors NI-35and NI-250. NI-250 is also known as Nogo (Chen et al Nature 403:434-39(2000); GrandPré et al, Nature 403: 439-444 (2000); Prinjha et al.,Nature 403: 383-84 (2000), all of which are herein incorporated byreference).

There are three isoforms of the Nogo protein, Nogo-A, -B, and -C, whichresult from alternative splicing or promoter usage. Nogo-A is thefull-length protein of 1192 amino acids and is expressed primarily inthe brain and optic nerve. Nogo-B, 373 amino acids, may correspond tothe NI-35 fraction of myelin preparation and is located in small amountsin the optic nerve. Nogo-C, 199 amino acids long, is found primarily inthe brain. Nogo-A and -B share the same common N-terminus of 172 aminoacids, while all three Nogo isoforms share a common C-terminal regionwhich shows approximately 70% similarity to the C-terminus of thereticulon (Rtn) family of proteins (GrandPré et al, supra). TheC-termini contain two hydrophobic transmembrane domains separated by a66 amino acid hydrophilic loop that protrudes from the cell surface.

Mapping Nogo neuronal growth inhibitory domains demonstrates that twodistinct sites play a role in preventing neurite outgrowth. The Nogo-Aprotein was shown to inhibit axonal growth in dorsal root ganglion (DRG)explants in vitro. Fine mapping of Nogo-A by Chen et al, (supra)demonstrates that the amino terminal portion, known as Amino-Nogo,inhibits neurite outgrowth in culture. The 66 amino acid linker ofNogo-C has inhibitory properties as well, inhibiting growth coneformation and inducing growth cone collapse in chick DRG neurons invitro (GrandPré et al supra). Further mapping of Nogo-66 revealed thatresidues 33-55 of the extracellular sequence are responsible for thegrowth cone inhibition (GrandPré et al supra).

The receptor for the Nogo-66 peptide was identified by Fournier et al.by using a Nogo-66-alkaline phosphatase fusion protein (Nogo-AP) whichwas shown to bind with high affinity to chick DRG axons (Fournier, etal., supra). The Nogo-66 receptor (NgR) is 473 amino acids, contains asignal sequence followed by eight leucine rich repeat (LRR) domains, anLRR flanking carboxy-terminal (LRRCT) domain that is cysteine-rich, aunique region, and a C-terminal glycophosphtidyl inositol (GPI)anchoring sequence (Fournier, et al., supra). The NgR mRNA is primarilyexpressed in the brain. Cleavage of NgR from the axonal cell surfacerenders neurons insensitive to Nogo-66. Furthermore, neurons that do notexpress NgR are insensitive to Nogo-66-induced growth cone collapse.However, expression of recombinant NgR in these cells renders axonalgrowth cones sensitive to Nogo-66-induced collapse, indicating that NgRfacilitates Nogo activity in neurons (Fournier, et al., supra).

Administration of antibodies generated against the NI-250 myelinfraction (IN-1; Caroni and Schwab, Neuron 1:85-96 (1988), hereinincorporated by reference) neutralizes the effects of NI-35/250 inculture and permits axon fibers extension in contrast to untreatedcells. IN-1 antibodies also improve the motor capabilities of adult ratsafter spinal cord injury (Bregman et al., Nature 378:498-501 (1995);Merkler et al., J. Neuroscience 27: 3665-73 (2001), all of which areherein incorporated by reference). These results indicate that Nogo is amajor factor in inhibiting CNS axonal regeneration and that blockingNogo activity can be an effective measure in restoring axonal functionafter spinal cord trauma.

Thus, there exists a need in the art to identify materials and methodsto modulate growth cone collapse and axonal regeneration. Identificationand development of such agents provides therapeutic compositions andmethods of treatment for neurological conditions such as spinal cordinjury, cranial or cerebral trauma, stroke, and demyelinating diseases.

The NgRHy polypeptide of SEQ ID NO: 420 is an approximately 420 aminoacid transmembrane protein with a predicted molecular mass ofapproximately 46 kDa unglycosylated. Protein database searches with theBLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altschul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 420 is homologous tohuman NgR.

FIG. 32 shows a schematic diagram illustrating the major structuralfeatures of the Nogo-receptor, NgR, and the Nogo-receptor homolog,NgRHy.

FIG. 33 shows the BLASTP amino acid sequence alignment between theprotein encoded by SEQ ID NO: 419 (i.e. SEQ ID NO: 420), NgRHy, and thehuman NgR (SEQ ID NO: 440), indicating that the two sequences share 48%identity over 358 amino acids of SEQ ID NO: 420 and 60% similarity overthe same 358 amino acids of SEQ ID NO: 420, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

A predicted approximately 16 residue signal peptide is encoded fromapproximately residue 1 through residue 30 of SEQ ID NO: 420 (SEQ ID NO:422). The extracellular portion (SEQ ID NO: 439) is useful on its own.This can be confirmed by expression in mammalian cells and sequencing ofthe cleaved product. The signal peptide region was predicted using theNeural Network SignalP V1.1 program (from Center for Biological SequenceAnalysis, The Technical University of Denmark). One of skill in the artwill recognize that the actual cleavage site may be different than thatpredicted by the computer program.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol. 6:219-235 (1999), herein incorporated byreference), NgRHy is expected to have five leucine-rich repeat (LRR)domains at residues 130-144 of SEQ ID NO: 420 (SEQ ID NO: 423), residues154-168 of SEQ ID NO: 420 (SEQ ID NO: 424), residues 157-171 of SEQ IDNO: 420 (SEQ ID NO: 425), residues 178-192 of SEQ ID NO: 420 (SEQ ID NO:426), and residues 250-264 of SEQ ID NO: 420 (SEQ ID NO: 427) domain asshown Table 16, wherein A=Alanine, C=Cysteine, D=Aspartic Acid,E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine,K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine:TABLE 16 Amino acid sequence SEQ Signature (start and ID NO: p-valueIdentification No. end position) 423 5.345e−08 PR00019A LERLQSLHLYRCQLS(130-144) 424 8.448e−08 PR00019B LVSLQYLYLQENSLH (154-168) 425 4.545e−08PR00019A LQYLYLQENSLLHLQ (157-171) 426 2.552e−08 PR00019BLANLSHLFLHGNRLR (178-192) 427 8.448e−08 PR00019B LPSLEFLRLNANPWA(250-264)

Using hmmpfam software (Washington University School of Medicine, St.Louis, Mo.), NgRHy was determined to have eight leucine-rich repeat(LRR) domain and a leucine-rich region-associated C-terminal (LRRCT)domain as shown in Table 17, wherein A=Alanine, C=Cysteine, D=AsparticAcid, E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine,I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine,P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine,W=Tryptophan, Y=Tyrosine: TABLE 17 SEQ Amino acid sequence ID (start andNO: Domain Score e-value end position) 428 Leucine-rich 2.7 2.4e+02STQRLFQNNLIRTLRPGTF repeat GS (42-63) 429 Leucine-rich 20.0 0.057NLLTLWLFSNNLSTIYPG repeat TFRHLQ (64-87) 430 Leucine-rich 22.6 0.0095ALEELDLGDNRHLRSLEP repeat DTFQGLE (88-112) 431 Leucine-rich 23.9 0.0037RLQSLHLYRCQLSSLPGN repeat IFRGLV (113-136) 432 Leucine-rich 18.3 0.18SLQYLYLQENSLLHLQD repeat DLFADLA (137-160) 433 Leucine-rich 15.9 0.97NLSHLFLHFNRLRLLTEH repeat VFRGLG (161-184) 434 Leucine-rich 16.5 0.62SLDFLLLHGNRLQGVHR repeat AAFRGLS (185-208) 435 Leucine-rich 23.1 0.0066RLTILYLFNNSLASLPGEA repeat LADLP (209-232) 436 Leucine-rich 38.9 1.2e−07NPWACDCRARPLWAWF repeat- QRARVSSSDVTCATPPER associated QGRDLRALREADFQACPC-terminal (242-292) domain

Using the Kyte-Doolittle hydrophobicity prediction algorithm (J. Mol.Biol., 157:105-131 (1982), incorporated herein by reference), NgRHy ispredicted to have a transmembrane domain at residues 382-396 (SEQ ID NO:437):LSAGLPSPLLCLLLL

-   -   wherein A-Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,        F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,        L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,        R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan,        Y=Tyrosine. Removal of the transmembrane domain renders a        soluble fragment that can be used to inhibit NgRHy and/or NgR        activity and is designated as SEQ ID NO: 438.

In particular, the NgRHy polypeptides and polynucleotides of theinvention may be used in the treatment of spinal cord injury, cranial orcerebral trauma, stroke, and demyelinating diseases.

The activity of an NgRHy polypeptide of the invention may manifest asmodulating neural growth activity, such as stimulation of neuriteoutgrowth, stimulation of neural cell proliferation, regeneration ofnerve and brain tissue, a soluble form of NgRHy can act as a competitiveinhibitor to block NgRHy thereby stimulating axonal growth,alternatively, NgRHy can act as a decoy receptor to modulate, i.e.stimulate or inhibit, axonal growth. The mechanism underlying theparticular condition or pathology will dictate whether NgRHypolypeptides, binding partners thereof, or inhibitors thereof would bebeneficial to the subject in need of treatment.

The present invention provides methods for modifying, such as inducingor inhibiting, proliferation of neural cells and for regeneration ofnerve and brain tissue, which comprise administering a composition ofNgRHy polypeptides, disclosed in the present invention. Such proteins ofthe present invention may be used to treat central and peripheralnervous system disorders, neuropathies, and lesions, as well asmechanical and traumatic disorders, which involve degeneration, death ortrauma to neural cells or nerve tissue. More specifically, a protein maybe used in the treatment of diseases of the peripheral nervous system,such as peripheral nerve injuries, peripheral neuropathy, and localizedneuropathies, and central nervous system diseases, such as Alzheimer'sdisease, Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome. Further conditions which may betreated in accordance with the present invention include mechanical andtraumatic disorders, such as spinal cord injuries, head trauma, andcerebrovascular diseases including stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a protein of the invention.

NgRHy polypeptides are used to produce antibodies that will bind toNgRHy and/or NgR, thereby inhibiting NgRHy and/or NgR activity.Inhibition of either receptor will block Nogo-induced neurite growthinhibition and can be an effective therapeutic to restore axonalfunction after injury or disease.

The soluble ectodomain of NgRHy is used as a competitive inhibitor tobind to and/or block the activity of NgRHy or NgR thereby renderingcells insenstitive to Nogo protein inhibition of axonal growth.

NgRHy inhibits Nogo-dependent signaling by acting as a decoy receptor.Binding of Nogo proteins and/or other ligands for NgR and NgRHy toectopically expressed NgRHy can result in decreased binding of saidligands to NgR thereby reducing the effect of Nogo signaling on axonalgrowth.

Antibodies raised agains the NgRHy polypeptide or fragment thereof, canbe used as a therapeutic for treatment of neurological conditions suchas spinal cord injury, cranial or cerebral trauma, stroke, anddemyelinating diseases. Anti-NgRHy antibodies can inhibit the activityof either NgRHy or NgR by blocking access, either by stericallyinhibiting binding of the ligand or by changing the conformation of thereceptor such that ligand binding does not occur or that the receptor isunable to activate downstream signaling molecules even if the ligand isbound.

4.4 Scavenger Receptor-Like Polypeptide

Macrophages actively uptake a wide range of molecules includingproteins, bacteria and viral particles, apoptotic cells and red bloodcells, and low density lipoproteins (LDLs) (Yamada et al., Cell MolecLife Sc 54:628-640 (1998) herein incorporated by reference). Thescavenger receptors were first reported as receptors for oxidized andacetyl-LDLs. From cross-competition experiments it has become clear thatmacrophages and other cells express several classes of scavengerreceptors. These receptors include type I and type II class A receptors,CD36 and SR-B1 class B receptors and CD68 and LOX-1 class C receptorsthat are distinct from the receptors for plasma LDLs. Atherosclerosisbegins when lipoproteins accumulate in the arterial intima and becomechemically modified thus initiating local vessel wall inflammation. Thisbrings in monocytes-derived macrophages which avidly take up themodified lipids, becoming fat-laden “foam” cells which reside in thevessel wall and exacerbate the local inflammation.

Class A type I and II macrophage scavenger receptors are trimericproteins of about 220-250 kDa with an amino-terminal collagenous domainthat is essential for ligand binding. Type I receptors have a scavengerreceptor Cysteine-rich domain (SRCR) while type II receptors do not.Receptors containing the SRCR domain bind immunoglobulin domaincontaining proteins and may serve as adhesion receptors. The collagendomains of these receptors have Gly-X-Y repeats and form a triplehelical structure. The modified LDL binding site resides at the carboxyterminus of the collagen domain in a stretch of basic amino acidresidues. The cytoplasmic domain is essential for cell surfaceexpression and receptor endocytosis.

Type I and II receptors are expressed on all tissue macrophages. Theyare also expressed in brain in the perivascular macrophages called MATOcells, and endothelial cells of the liver, the adrenal gland and lymphnodes. Cytokines and other growth factors are known to modulatescavenger receptor expression. Type I and type II receptors bind andendocytose multiple ligands including acetyl-LDL, advanced glycation endproducts (AGE), and apoptotic cells. They also bind bacterialendotoxins, gram-positive bacteria and recognize lipoteichoic acid. Thebinding of endotoxins does not lead to endotoxin signaling and thus maybe a way of getting rid of excess endotoxins. They also recognizeListeria and herpes simplex virus. Type I and type II scavengerreceptors also mediate cell adhesion and may assist in developing robustimmune response. In the brain, accumulation of the scavenged materialsresults in the formation of foam cells similar to that found withatherosclerosis and contributes to narrowing of the lumen of thearterioles in the cortex.

Thus, the scavenger receptor-like polypeptides and polynucleotides ofthe invention may be used in the treatment of atherosclerosis, disorderscaused by the accumulation of denatured materials and cellular debris,bacterial and viral infections, inflammation, strengthening of immuneresponse, and Alzheimer's disease.

The scavenger receptor-like polypeptide of SEQ ID NO: 444 is anapproximately 495-amino acid protein with a predicted molecular mass ofapproximately 54 kDa unglycosylated.

Protein database searches with the BLASTX algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 444 is homologous to macrophage scavenger receptors.

FIG. 34 shows the BLASTX amino acid sequence alignment between theprotein encoded by SEQ ID NO: 443 (i.e. SEQ ID NO: 444) scavengerreceptor-like polypeptide and mouse macrophage scavenger receptor type I(SEQ ID NO: 481), indicating that the two sequences share 57% similarityover a 335 amino acid residue region of SEQ ID NO: 444 and 40% identityover the same 335 amino acid residues of SEQ ID NO: 444. The resultsalso indicate that the two sequences share 49% similarity over adistinct 77 amino acid residue region of SEQ ID NO: 444 and 31% identityover the same 77 amino acid residues of SEQ ID NO: 444, whereinA=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.26:320-322 (1998) herein incorporated by reference) SEQ ID NO: 444 wasexamined for domains with homology to certain peptide domains. Table 18shows the SEQ ID NO: of the Pfam domain within SEQ ID NO: 444, the nameof the Pfam model found, the description, the e-value, Pfam score,number of repeats, and position of the domain within SEQ ID NO: 444 forthe identified model within the sequence as follows wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine: TABLE 18 SEQ ID Re- NO: ModelDescription e-value Score peats Position 482 SRCR Scavenger 2e−33 172.01 396-493 receptor cysteine-rich domain 483 Collagen Collagen triple9.1e−13   55.8 1 315-374 helix repeatFurther description of the Pfam models can be found athttp://pfam.wustl.edu/.

A predicted approximately twenty-one residue transmembrane domain isencoded from approximately residue 61 through residue 81 of SEQ ID NO:444 (SEQ ID NO: 484). The protein (SEQ ID NO: 444) lacking itstransmembrane portion may be useful on its own. This can be confirmed byexpression in mammalian cells. Presence of the transmembrane region wasdetected using the TMpred program (Hofmann and Stoffel, Biol. Chem.374:166 (1993), herein incorporated by reference). One of skill in theart will recognize that the actual transmembrane region may be differentthan that predicted by the computer program.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol. 6:219-235 (1999), herein incorporated byreference), scavenger receptor-like polypeptide (SEQ ID NO: 444) isexpected to have fourteen C1q domain proteins signatures as shown inTable 18. Using eMATRIX software package (Stanford University, Stanford,Calif.) (Wu et al., J. Comp. Biol. 6:219-235 (1999), herein incorporatedby reference), scavenger receptor-like polypeptide (SEQ ID NO: 444) isalso expected to have sixteen Speract receptor repeat proteins domainproteins signatures as shown in Table 19. Using eMATRIX software package(Stanford University, Stanford, Calif.) (Wu et al., J. Comp. Biol.6:219-235 (1999), herein incorporated by reference), scavengerreceptor-like polypeptide (SEQ ID NO: 444) is also expected to have fiveSperact receptor signatures as shown in Table 19. The domainscorresponding to SEQ ID NO: 445-479 are as follows: TABLE 19 SEQ IDDatabase NO p-Value Entry ID Description Position* 447 3.189e−13 BL01113C1q domain proteins 324-350 451 5.295e−11 BL01113 C1q domain proteins306-332 453 1.383e−10 BL01113 C1q domain proteins 333-359 456 2.149e−10BL01113 C1q domain proteins 318-344 457 2.915e−10 BL01113 C1q domainproteins 321-347 458 7.128e−10 BL01113 C1q domain proteins 327-353 4601.692e−09 BL01113 C1q domain proteins 342-368 463 4.115e−09 BL01113 C1qdomain proteins 312-338 464 5.673e−09 BL01113 C1q domain proteins315-341 470 7.517e−08 BL01113 C1q domain proteins 330-356 471 1.000e−07BL01113 C1q domain proteins 309-335 472 1.415e−07 BL01113 C1q domainproteins 354-380 474 3.077e−07 BL01113 C1q domain proteins 339-365 4796.123e−07 BL01113 C1q domain proteins 303-329 445 8.333e−39 BL00420Speract receptor repeat proteins domain proteins 397-451 448 9.100e−13BL00420 Speract receptor repeat proteins domain proteins 482-492 4509.135e−12 BL00420 Speract receptor repeat proteins domain proteins309-337 452 7.382e−11 BL00420 Speract receptor repeat proteins domainproteins 324-352 455 1.885e−10 BL00420 Speract receptor repeat proteinsdomain proteins 348-376 459 7.639e−10 BL00420 Speract receptor repeatproteins domain proteins 306-334 461 2.246e−09 BL00420 Speract receptorrepeat proteins domain proteins 321-349 465 4.423e−08 BL00420 Speractreceptor repeat proteins domain proteins 336-364 467 5.183e−08 BL00420Speract receptor repeat proteins domain proteins 312-340 468 5.310e−08BL00420 Speract receptor repeat proteins domain proteins 339-367 4697.338e−08 BL00420 Speract receptor repeat proteins domain proteins327-355 473 3.077e−07 BL00420 Speract receptor repeat proteins domainproteins 315-343 475 4.462e−07 BL00420 Speract receptor repeat proteinsdomain proteins 351-379 476 5.615e−07 BL00420 Speract receptor repeatproteins domain proteins 333-361 477 5.962e−07 BL00420 Speract receptorrepeat proteins domain proteins 342-370 478 5.962e−07 BL00420 Speractreceptor repeat proteins domain proteins 345-373 446 8.054e−16 PR00258SPERACT RECEPTOR SIGNATURE 393-409 449 1.509e−12 PR00258 SPERACTRECEPTOR SIGNATURE 412-423 454 1.833e−10 PR00258 SPERACT RECEPTORSIGNATURE 481-493 462 3.667e−09 PR00258 SPERACT RECEPTOR SIGNATURE427-437 466 4.971e−08 PR00258 SPERACT RECEPTOR SIGNATURE 458-472*Position of signature in amino acid sequence (i.e. SEQ ID NO: 444)

In particular, the scavenger receptor-like polypeptides andpolynucleotides of the invention may be used in the treatment ofatherosclerosis, disorders caused by the accumulation of denaturedmaterials and cellular debris, bacterial and viral infections,inflammation, strengthening of the immune response, and Alzheimer'sdisease.

4.5 Neural Immunoglobulin Cell Adhesion Molecule-Like (Neural IgCAM)Polypeptides

The establishment of neural connections during development is a highlydynamic process. A key aspect of this process is the regulation of axongrowth, which is mediated by a variety of chemotropic factors (Skaper,et al., Prog. Neurobiol. 56:593-608 (2001), incorporated herein byreference). Chemotropism, which determines the direction of axonalgrowth, results from the concerted action of chemoattractant andchemorepellent cues (Yu and Bargmann, Nat. Neurosci. 4 (Suppl.):1169-1176 (2001), incorporated herein by reference). Growth cones, theleading edge of the axons, encounter and detect these guiding cues alongtheir trajectories in the form of gradients of diffusible factors,necessary for long-range guidance (Zheng and Kuffler, J. Neurobiol.42:212-219 (2000), incorporated herein by reference), extracellularmatrix-associated molecules, required for both short- and long-rangeregulation (Hynds and Snow, Exp. Neurol. 160:244-255 (1999),incorporated herein by reference; Skaper et al., 2001. supra), andmembrane-bound molecules, necessary for short-range regulation (He andMeini, Mol. Cell. Neurosci. 19:18-31 (2002), incorporated herein byreference). It is believed that the inability of mature neurons toregenerate appropriate connections following injury or trauma is in partmediated by chemorepellent molecules present along axonal tracts(Fawcett, Cell Tissue Res. 290:371-377 (1997), incorporated herein byreference). During neural development, both membrane-bound and solubleproteins regulate axonal growth towards their targets. Integrins,cadherins and neural cell adhesion molecules (NCAMs) generally promoteneurite outgrowth. Immunoglobulin superfamily members like L1 and NCAMare widely expressed and promote outgrowth of most neurons (Gil et al.,J. Neurosci. 18:9312-9325 (1998), incorporated herein by reference).

Signals generated following neural IgCAM binding lead to alterations incellular signaling and morphology affecting cell migration,proliferation, and differentiation. Subfamilies of neural IgCAMs arecategorized according to the number of immunoglobulin (Ig) domains andfibronectin repeats, as well as the mode of attachment to the cellsurface (either a transmembrane domain or a glycophosphatidyl inositollinkage), and the presence of a catalytic cytoplasmic domain (reviewedin Crossin and Krushel, Dev. Dyn. 218:260-279 (2000), hereinincorporated by reference). A number of studies have correlated NCAMexpression with the establishment of learning and memory (reviewed inRose, Trends Neurosci. 18:502-506 (1995), herein incorporated byreference) as well as in schizophrenia (Poltorak et al., Brain Res.751:152-154 (1997), herein incorporated by reference). Specific tyrosinekinases have been implicated in the effects of neural IgCAMs in neuriteoutgrowth (reviewed in Doherty and Walsh, Curr. Opin. Neurobiol. 4:49-55(1994), herein incorporated by reference). Specifically, the fibroblastgrowth factor (FGF) receptor has been shown to be stimulated byinteractions with neural IgCAMs via a “CAM homology domain” in the FGFreceptor (Williams et al., Neuron 13:583-594 (1994); Williams et al., J.Cell Sci. 108:3523-3530 (1995), herein incorporated by reference).Additionally, nonreceptor tyrosine kinases, such as ERK1 and ERK2 havebeen implicated in signaling pathways associated with neural IgCAM inneurite outgrowth (Schmid et al., J. Neurobiol. 38:542-558 (1999),herein incorporated by reference).

Five exemplary neural IgCAM sequences of the invention are describedbelow: amino acid sequence SEQ ID NO: 487 (and encoding nucleotidesequence SEQ ID NO: 486), amino acid SEQ ID NO: 505 (and encodingnucleotide sequence SEQ ID NO: 504), amino acid sequence SEQ ID NO: 516(and encoding nucleotide sequence SEQ ID NO: 515), amino acid sequenceSEQ ID NO: 528 (and encoding nucleotide sequence SEQ ID NO: 527), andamino acid sequence SEQ ID NO: 542 (and encoding nucleotide sequence SEQID NO: 541).

The first neural IgCAM-like polypeptide of SEQ ID NO: 487 is anapproximately 1029-amino acid protein with a predicted molecular mass ofapproximately 1113-kDa unglycosylated. The initial methionine starts atposition 178 of SEQ ID NO: 486 and the putative stop codon begins atposition 3262 of SEQ ID NO: 486. A signal peptide of 18 residues ispredicted from approximately residue 1 to residue 18 of SEQ ID NO: 487(i.e. SEQ ID NO: 489). The extracellular portion is useful on its own.This can be confirmed by expression in mammalian cells and sequencing ofthe cleaved product. The signal peptide region was predicted using theNeural Network SignalP V1.1 program (Nielsen et al, Int. J. Neural Syst.8:581-599 (1997)). One of skill in the art will recognize that theactual cleavage site may be different than that predicted by thecomputer program.

Using the TMpred program (Hofmann and Stoffel, Biol. Chem. 374:166(1993), herein incorporated by reference), SEQ ID NO: 487 is predictedto have a transmembrane domain at approximately residue 904 to residue920. Removal of the transmembrane domain renders soluble fragments thatcan be used to inhibit receptor activity. An exemplary extracellulardomain spans approximately residue 19 to residue 903 of SEQ ID NO: 487(i.e. SEQ ID NO: 501).

Protein database searches with the BLASTP algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 487 is homologous to murine PANG, a neuronal CAM (SEQ ID NO:502).

FIG. 35 shows the BLASTP amino acid sequence alignment between theprotein derived from SEQ ID NO: 486 (i.e. SEQ ID NO: 487) and murinePANG amino acids 1-1028 of SEQ ID NO: 502, indicating that the twosequences share 93% similarity over 1028 amino acid residues of SEQ IDNO: 487 and 87% identity over the same 1028 amino acid residues of SEQID NO: 487, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are represented as dashes.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference), neural IgCAM-likepolypeptide of SEQ ID NO: 487 is predicted to contain fiveimmunoglobulin (Ig) domains and four fibronectin type III (FN3) domainsas shown in Table 20, wherein A=Alanine, C=Cysteine, D=Aspartic Acid,E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine,K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Further description of the Pfam models can be found athttp://pfam.wustl.edu/. TABLE 20 Amino SEQ acid sequence encoded ID(start and end amino NO: Domain Score e-value acid position 491 Igdomain 29.4 1.4e−07 EKKVKLNCEVKGNPkPhYRW KLNGTDVDTGMDFRYSVVEGSLLINNPNKTQDAGTYQCTA (43-102) 492 Ig domain 23.8 8.2e−06GQGVVLLCGPPPHSGELSYA WIFNEYPSFVEEDSRRFVSQE TGHLYISKVEPSDVGNYTCVV(137-198) 493 Ig domain 38.4 2.3e−10 GSTVKLECFALGNPIPQINWRRSDGLPFSSKIKLRKFSGVLE IPNFQQEDAGSYECIA (242-299) 494 Ig domain 32.51.6e−08 GSLVSLDCKPRASPRALSSWK KGDVSVQEHERISLLNDGGLK IANVTKADAGTYTCMA(424-481) 495 Ig domain 26.2 1.4e−06 ESVILPCQVQHDPLLDIIFTWYFNGALADFKKDGSHFEKVGG SSSGDLMLRNIQLKHSGKYVC MV (514-579) 496 FN3 domain83.0 6.0e−21 PGPPENVKVDEITDTTAQLSW KEGKDNHSPVISYSIQARTPFSVGWQTVTTVPEVIDGKTHTA TVVELNPWVEYEFRVVASNKI GGGEPS (598-687) 497 FN3domain 30.7 3.4e−05 PEVPPSEVNGGGGSRSELVIT WDPVPEELQNGEGFGYVVAFRPLGVTTWIQTVVTSPDTPRY VFRNESIYPYSPYEVKVGVYN NKGEGPFS (700-790) 498 FN3domain 61.9 1.4e−14 PTVAPSQVSANSLSSSEIEVS WNTIPWKLSNGHLLGYEVRYWNGGGPTVAPSQVSANSLSSSE IEVSWNTIPWKLSNGHLLGYE VRYWNGGG (802-891) 499 FN3domain 36.7 5.2e−17 PSQPPGNVVWNATDTKVLLN WEQVKAMENESEVTGYKVFYRTSSQNNVQVLNTNKTSAELV LPIKEDYIIEVKATTDGGDGT SS (903-986)

The second neural IgCAM-like polypeptide of SEQ ID NO: 505 is anapproximately 231-amino acid protein with a predicted molecular mass ofapproximately 25-kDa unglycosylated. The initial methionine starts atposition 17 of SEQ ID NO: 504 and the putative stop codon begins atposition 707 of SEQ ID NO: 504. A signal peptide of 20 residues ispredicted from approximately residue 1 to residue 20 of SEQ ID NO: 505(i.e. SEQ ID NO: 507). The extracellular portion is useful on its own.This can be confirmed by expression in mammalian cells and sequencing ofthe cleaved product. The signal peptide region was predicted using theNeural Network SignalP V1.1 program (Nielsen et al, Int. J. Neural Syst.8:581-599 (1997)). One of skill in the art will recognize that theactual cleavage site may be different than that predicted by thecomputer program.

Using the TMpred program (Hofmann and Stoffel, Biol. Chem. 374:166(1993), herein incorporated by reference), SEQ ID NO: 505 is predictedto have a transmembrane domain at approximately residue 213 to residue230. Removal of the transmembrane domain renders soluble fragments thatcan be used to inhibit receptor activity. An exemplary extracellulardomain spans approximately residue 21 to residue 212 of SEQ ID NO: 505(i.e. SEQ ID NO: 512).

Protein database searches with the BLASTP algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 505 is homologous to bovine NCAM-140 precursor (SEQ ID NO:513).

FIG. 36 shows the BLASTP amino acid sequence alignment between theprotein derived from SEQ ID NO: 504 (i.e. SEQ ID NO: 505) and bovineNCAM-140 precursor amino acids 343-528 of SEQ ID NO: 513, indicatingthat the two sequences share 45% similarity over 191 amino acid residuesof SEQ ID NO: 505 and 29% identity over the same 191 amino acid residuesof SEQ ID NO: 505, wherein A=Alanine, C=Cysteine, D=Aspartic Acid,E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine,K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are represented as dashes.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference), neural IgCAM-likepolypeptide of SEQ ID NO: 505 is predicted to contain two immunoglobulin(Ig) domains as shown in Table 21, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Further description of the Pfammodels can be found at http://pfam.wustl.edu/. TABLE 21 Amino SEQ acidsequence encoded ID (start and end amino NO: Domain Score e-value acidposition 509 Ig domain 13.1 0.017 GSQASLICAVQNHTREEELLWYREEGRVDLKSGNKINSSSVC VSSISENDNGISFTCRL (39-97) 510 Ig domain 43.17.3e−12 GSNLKLVCNVKANPQAQMM WYKNSSLLDLEKSRHQIQQTS ESFQLSITKVEKPDNGTYSCMA (128-189)

The third neural IgCAM-like polypeptide, SEQ ID NO: 541, is a variant ofSEQ ID NO: 504. SEQ ID NO: 541 contains a 10 bp insertion betweennucleotides 701 and 702 of SEQ ID NO: 504. The neural IgCAM-likepolypeptide of SEQ ID NO: 541 (i.e. SEQ ID NO: 542) is an approximately256 amino acid protein with a prediceted molecular mass of approximately28 kDa unglycosylated. The initial methionine starts at position 17 ofSEQ ID NO: 541 and the putative stop codon begins at position 788 of SEQID NO: 541. A signal peptide of 20 residues is predicted fromapproximately residue 1 to residue 20 of SEQ ID NO: 542 (i.e. SEQ ID NO:507). The extracellular portion is useful on its own. This can beconfirmed by expression in mammalian cells and sequencing of the cleavedproduct. The signal peptide region was predicted using the NeuralNetwork SignalP V1.1 program (Nielsen et al, Int. J. Neural Syst.8:581-599 (1997)). One of skill in the art will recognize that theactual cleavage site may be different than that predicted by thecomputer program.

Using the TMpred program (Hofmann and Stoffel, Biol. Chem. 374:166(1993), herein incorporated by reference), SEQ ID NO: 542 is predictedto have a transmembrane domain at approximately residue 217 to residue236 (i.e. SEQ ID NO: 545). Removal of the transmembrane domain renderssoluble fragments that can be used to inhibit receptor activity. Anexemplary extracellular domain spans approximately 21 to residue 216 ofSEQ ID NO: 542 (i.e. SEQ ID NO: 546).

Protein database searches with the BLASTP algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 542 is homologous to bovine NCAM-140 precursor (SEQ ID NO:513).

FIG. 37 shows a multiple amino acid sequence alignment between neuralIgCAM-like polypeptide SEQ ID NO: 505, neural IgCAM-like polypeptide SEQID NO: 542 and bovine NCAM-140 precursor (SEQ ID NO: 513), whereinA=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are represented as dashes (-), asterisks (*) represent identicalamino acids, colons (:) represent conservative substitutions, andperiods (.) represent semi-conservative substitutions.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference), neural IgCAM-likepolypeptide of SEQ ID NO: 542 is predicted to contain two immunoglobulin(Ig) domains as shown in Table 22, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Further description of the Pfammodels can be found at http://pfam.wustl.edu/. TABLE 22 Amino SEQ acidsequence encoded ID (start and end amino NO: Domain Score e-value acidposition 509 Ig domain 15.6 0.012 GSQASLICAVQNHTREEELLWYREEGRVDLKSGNKINSSSVC VSSISENDNGISFTCRL (39-97) 510 Ig domain 44.61.1e−10 GSNLKLVCNVKANPQAQMM WYKNSSLLDLEKSRHQIQQTS ESFQLSITKVEKPDNGTYSCMA (128-189)

The fourth neural IgCAM-like polypeptide of SEQ ID NO: 516 is anapproximately 674-amino acid protein with a predicted molecular mass ofapproximately 74-kDa unglycosylated. The initial methionine starts atposition 1 of SEQ ID NO: 516 and the putative stop codon begins atposition 2000 of SEQ ID NO: 515. A signal peptide of 32 residues ispredicted from approximately residue 1 to residue 32 of SEQ ID NO: 516(i.e. SEQ ID NO: 518). The extracellular portion is useful on its own.This can be confirmed by expression in mammalian cells and sequencing ofthe cleaved product. The signal peptide region was predicted using theNeural Network SignalP V1.1 program (Nielsen et al, Int. J. Neural Syst.8:581-599 (1997)). One of skill in the art will recognize that theactual cleavage site may be different than that predicted by thecomputer program.

Protein database searches with the BLASTP algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 516 is homologous to murine CAM, DDM36 (SEQ ID NO: 525).

FIG. 38 shows the BLASTP amino acid sequence alignment between theprotein derived from SEQ ID NO: 515 (i.e. SEQ ID NO: 514) and murineDDM36 amino acids 136-671 of SEQ ID NO: 525, indicating that the twosequences share 60% similarity over 540 amino acid residues of SEQ IDNO: 516 and 43% identity over the same 540 amino acid residues of SEQ IDNO: 516, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are represented as dashes.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference), neural IgCAM-likepolypeptide of SEQ ID NO: 516 is predicted to contain threeimmunoglobulin (Ig) and two fibronectin type III (FN3) domains as shownin Table 23, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Further description of the Pfam models can be found athttp://pfam.wustl.edu/. TABLE 23 Amino SEQ acid sequence encoded ID(start and end amino NO: Domain Score e-value acid position 520 Igdomain 28.2 3.6e−07 GGVARFACKISSHPPAVITWE FNRTTLPMTMDRITALPTGVLQIYDVSQRDSGNYRCIA (124-182) 521 Ig domain 25.4 2.5e−06HQTVVLECMATGNPKPIISWS RLDHKSIDVFNTRVLGNGNL MISDVRLQHAGVYVCRA (224-281)522 Ig domain 31.4 3.6e−08 AGTARFVCQAEGIPSPKMSWL KNGRKIHSNGRIKMYNSKLVINQIIPEDDAIYQCMA (316-372) 523 FN3 domain 60.2 4.3e−14PSAPYNVHAETMSSSAILLAW ERPLYNSDKVIAYSVHYMKA EGLNNEEYQVVIGNDTTHYIIDDLEPASNYTFYIVAYMPMG ASQMS (394-480) 524 FN3 domain 62.4 9.5e−15PLRPPEISLTSRSPTDILISW LPIPAKYRRGQVVLYRLSFRL STENSIQVLELPGTTHEYLLEGLKYPDSVYLVRITAATRVGL GESS (492-578)

The fifth neural IgCAM-like polypeptide of SEQ ID NO: 528 is anapproximately 1045-amino acid protein with a predicted molecular mass ofapproximately 115-kDa unglycosylated. The initial methionine starts atposition 117 of SEQ ID NO: 527 and the putative stop codon begins atposition 3249 of SEQ ID NO: 527. A signal peptide of 18 residues ispredicted from approximately residue 1 to residue 18 of SEQ ID NO: 528(i.e. SEQ ID NO: 530). The extracellular portion is useful on its own.This can be confirmed by expression in mammalian cells and sequencing ofthe cleaved product. The signal peptide region was predicted using theNeural Network SignalP V1.1 program (Nielsen et al, Int. J. Neural Syst.8:581-599 (1997)). One of skill in the art will recognize that theactual cleavage site may be different than that predicted by thecomputer program.

Using the TMpred program (Hofmann and Stoffel, Biol. Chem. 374:166(1993), herein incorporated by reference), SEQ ID NO: 528 is predictedto have a transmembrane domain at approximately residue 1023 to residue1040. Removal of the transmembrane domain renders soluble fragments thatcan be used to inhibit receptor activity. An exemplary extracellulardomain spans approximately residue 19 to residue 1022 of SEQ ID NO: 528(i.e. SEQ ID NO: 539).

Protein database searches with the BLASTP algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 528 is homologous to a rat CAM, BIG-2 precursor (SEQ ID NO:540).

FIG. 39 (A, B) shows the BLASTP amino acid sequence alignment betweenthe protein derived from SEQ ID NO: 527 (i.e. SEQ ID NO: 528) and ratBIG-2 precursor amino acids 5-1026 of SEQ ID NO: 540, indicating thatthe two sequences share 97% similarity over 1023 amino acid residues ofSEQ ID NO: 528 and 93% identity over the same 1023 amino acid residuesof SEQ ID NO: 528, wherein A=Alanine, C=Cysteine, D=Aspartic Acid,E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine,K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are represented as dashes.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference), neural IgCAM-likepolypeptide of SEQ ID NO: 528 is predicted to contain fourimmunoglobulin (Ig) and two fibronectin type III (FN3) domains as shownin Table 24, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Further description of the Pfam models can be found athttp://pfam.wustl.edu/. TABLE 24 Amino SEQ acid sequence encoded ID(start and end amino NO: Domain Score e-value acid position 532 Igdomain 30.1 8.7e−08 EKKVKLNCEVKGNPKPHIRW KLNGTDVDTGMDFRYSVVEGSLLINNPNKTQDAGTYQCTA (61-120) 533 Ig domain 36.5 9.1e−10GATVKLECFALGNPVPTIIWR RADGKPIARKARRHKSNGILE IPNFQQEDAGLYECVA (258-315)534 Ig domain 36.0 1.3e−09 GGEVVLECKPKASPKPVYTWK KGRDILKENERITISEDGNLRIINVTKSDAGSYTCIA (440-497) 535 Ig domain 26.5 1.1e−06GESIVLPCQVTHDHSLDIVFT WSFNGHLIDFDRDGDHEERVG GQDSAGDLMIRNIQLKHAGK YVCMV(530-596) 536 FN3 domain 73.2 5.4e−18 PGPPEAVTIDEITDTTAQLSWRPGPDNHSPITMYVIQARTPF SVGWQAVSTVPELIDGKTFTA TVVGLNPWVEYEFRTVAANVI GIGEPS(615-704) 537 FN3 domain 51.6 1.8e−11 PTKPPASIFARSLSATDIEVFWASPLEKNRGRIQGYEVKYWR HEDKEENARKIRTVGNQTSTK ITNLKGSVLYHLAVKAYNSAG TGPSS(819-907)

Neural IgCAMs, such as BIG-2, PANG, and NCAM-140 mediate the formation,maintenance, and plasticity of functional neuronal networks (Yoshihara,et al., J. Neurobiol., 28:51-69 (1995), herein incorporated byreference). These neural IgCAMs facilitate neurite extension promotingaxon growth and guidance (Connelly, et al., Proc. Natl. Acad. Sci. USA,91:1337-1341 (1994), herein incorporated by reference). Neural IgCAMsmediate interactions with the extracellular environment by binding toextracellular matrix proteins, such as NCAM-140 binding to heparansulfate proteoglycans (Prag, et al., J. Cell. Sci., 115:283-292 (2002),herein incorporated by reference). Neural IgCAMs are found predominantlyon neural cells, but are also found on muscle cells, NK cells, T cells,and transiently expressed on a variety of cells during embryogenesis.PANG is a neural glycoprotein that is found primarily in neuronal cells,but is also ectopically expressed on plasmacytoma cells indicating thatit may play a role in tumor metastasis as well as in axon guidance(Connelly, et al., 2001. supra).

The polypeptides of the invention are expected to have similaractivities as those listed above, and therefore would be involved inneural development, specifically neurite outgrowth, neural cellproliferation, as well as in learning, behavior, and memory.

The polypeptides, polynucleotides, antibodies and other compositions ofthe invention are expected to provide potential treatments for disordersinvolving, but not limited to cognition, memory and learning, mood,dementia (including without limitation Alzheimer's disease, dementiaassociated with Parkinson's disease, multi-infarct dementia and others),depression, anxiety (including without limitation manic-depressiveillness, obsessive-compulsive disorders, generalized anxiety andothers), different forms of epilepsy, schizophrenia and schizophrenaformdisorders (including without limitation schizoaffecto disorder),cerebral palsy and hypertension (see, e.g. U.S. Pat. No. 5,861,283,incorporated herein by reference). The polypeptides, polynucleotides,antibodies and other compositions of the invention may providetherapeutic compositions and methods of treatment for neurologicalconditions such as spinal cord injury, cranial or cerebral trauma,stroke, demyelinating diseases, and other neurodegenerative disordersincluding amyotrophic lateral sclerosis, progressive spinal muscularatrophy, progressive bulbar paralysis of childhood (Fazio-Londesyndrome), poliomyelitis and post polio syndrome, and hereditary motorsensory neuropathy (Charcot-Marie-Tooth Disease).

4.6 Growth Hormone-Like Polypeptides and Polynucleotides

Human growth hormone (hGH), also known as somatotropin, is a member of afamily of homologous hormones that include placental lactogens,prolactins, and other genetic and species variants of growth hormone(Nichol et al., Endocrine Reviews, 7:169 (1986), incorporated herein byreference). The hGH gene cluster is located on chromosome 17 andconsists of five highly conserved genes, hGH-N, hGH-V, hCS-L, hCS-A, andhCS-B. Human growth hormone-N is a 22,000-dalton hormone expressed inthe somatotrope and lactosomatotrope cells of the anterior pituitary.Human growth hormone-N exhibits a multitude of biological effects,including linear growth (somatogenesis), lactation, activation ofmacrophages, and insulin-like and diabetogenic effects, among others(Chawla, Annu. Rev. Med., 34:519 (1983), incorporated herein byreference; Edwards et al., Science, 39:769 (1988), incorporated hereinby reference; Isaksson et al., Annu. Rev. Physiol., 47:483 (1985),incorporated herein by reference; Thomer and Vance, J. Clin. Invest.,82:745 (1988), incorporated herein by reference; Hughes and Friesen,Annu. Rev. Physiol., 47:469 (1985), incorporated herein by reference).It promotes growth in the size of the limbs and internal organs.Hypersecretion of hGH causes giantism or acromegaly while its deficiencyin children promotes dwarfism.

The remaining four genes of the growth hormone family, hGH-V, hCS-L,hCS-A, and hCS-B, are expressed in the syncytiotrophoblastic layer ofthe mid- to late gestational placenta (Su et al., J. Biol. Chem., 275;11(2000), incorporated herein by reference). The hGH-V gene, also known asgrowth hormone-2, is a natural analog of hGH-N and is also potentsomatogen. Like hGH-N, it binds growth hormone binding protein,increases glucose oxidation, induces refractoriness to insulin-likestimulation and lipolysis in the presence of glucocorticoids.

The biological effects of hGH derive from the interaction between hGHand specific cellular receptors. These interactions activate signalingpathways which contribute to growth hormone-induced changes in enzymaticactivity, transport function, and gene expression that ultimatelyculminate in changes in growth and metabolism (Carter-Su et al., Annu.Rev. Physiol., 5:187 (1996), incorporated herein by reference).

Two exemplary growth hormone-like sequences of the invention aredisclosed below: amino acid sequence SEQ ID NO: 548 (and encodingnucleotide sequence SEQ ID NO: 549) and amino acid sequence SEQ ID NO:557 (and encoding nucleotide sequence SEQ ID NO: 556). The growthhormone-like polypeptide of SEQ ID NO: 548 is an approximately 173-aminoacid protein with a predicted molecular mass of approximately 19 kDaunglycosylated. The initial methionine starts at position 58 of SEQ IDNO: 547 and the putative stop codon begins at position 577 of SEQ ID NO:547. A signal peptide of twenty-six residues is predicted fromapproximately residue 1 to residue 26 of SEQ ID NO: 548. Theextracellular portion is useful on its own. This can be confirmed byexpression in mammalian cells and sequencing of the cleaved product. Thesignal peptide region was predicted using the Neural Network SignalPV1.1 program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)). Oneof skill in the art will recognize that the actual cleavage site may bedifferent than that predicted by the computer program.

Protein database searches with the BLASTP algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 548 is homologous to somatotropin/prolactin hormones.

FIG. 40 shows the BLASTP amino acid sequence alignment between growthhormone-like polypeptide SEQ ID NO: 548 and human chorionicsomatomammotropin hormone-like 1, isoform 3 precursor (SEQ ID NO: 554),indicating that the two sequences share 89% similarity over 77 aminoacid residues and 85% identity over the same 77 amino acid residues,wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

FIG. 41 shows the BLASTP amino acid sequence alignment between growthhormone-like polypeptide SEQ ID NO: 548 and human chorionicsomatomammotropin hormone-like 1, isoform 5 percursor (SEQ ID NO: 555),indicating that the two sequences share 100% identity over 63 amino acidresidues, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference) SEQ ID NO: 548 wasexamined for domains with homology to known conserved peptide domains.Table 25 shows the SEQ ID NO: of the Pfam domain, the name of the Pfammodel found, the description, the e-value, Pfam score, number ofrepeats, and position of the domain within SEQ ID NO: 548 for theidentified model within the sequence as follows: TABLE 25 SEQ ID Re- NO:Model Description E-value Score peats Position 550 hormone Somatotropin1.6e−17 48.2 1 9-57 hormone family

Using the eMATRIX software package (Stanford University, Stanford,Calif.) (Wu et al., J. Comp. Biol., 6:219-235 (1999), hereinincorporated by reference), the growth hormone-like polypeptide of SEQID NO: 548 was determined to have following the eMATRIX domain hits. Theresults in Table 26 describe: SEQ ID NO of the eMATRIX domain, thecorresponding p-value, subtype, Signature ID number, domain name, theamino acid sequence of the eMATRIX domain and the corresponding positionof the amino acids within SEQ ID NO: 548, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine TABLE 26 Amino acid sequence SEQencoded (start ID Signature and end amino NO p-value ID NO Name acidposition) 551 8.347e−11 BL00266A Somatotropin, LFKEAMLQAHRAHQ prolactinand LAIDTYQEFISSW related (35-61) hormones proteins

The second growth hormone-like polypeptide of SEQ ID NO: 557 is anapproximately 256-amino acid protein with a predicted molecular mass ofapproximately 28 kDa unglycosylated. The initial methionine starts atposition 58 of SEQ ID NO: 556 and the putative stop codon begins atposition 826 of SEQ ID NO: 556. A signal peptide of twenty-six residuesis predicted from approximately residue 1 to residue 26 of SEQ ID NO:557. The extracellular portion is useful on its own. This can beconfirmed by expression in mammalian cells and sequencing of the cleavedproduct. The signal peptide region was predicted using the NeuralNetwork SignalP V1.1 program (Nielsen et al, Int. J. Neural Syst.8:581-599 (1997)). One of skill in the art will recognize that theactual cleavage site may be different than that predicted by thecomputer program.

Protein database searches with the BLASTP algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 557 is homologous to somatotropin/prolactin hormones.

FIG. 42 shows the BLASTP amino acid sequence alignment between growthhormone-like polypeptide (SEQ ID NO: 557) and human chorionicsomatomammotropin hormone 1, isoform 2 precursor (SEQ ID NO: 568),indicating that the two sequences share 94% similarity over 256 aminoacid residues and 92% identity over the same 256 amino acid residues,wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

FIG. 43 shows the BLASTP amino acid sequence alignment between growthhormone-like polypeptide (SEQ ID NO: 557) and human growth hormone 2,isoform 2 precursor (SEQ ID NO: 569), indicating that the two sequencesshare 84% similarity over 256 amino acid residues and 79% identity overthe same 256 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference) SEQ ID NO: 557 wasexamined for domains with homology to known conserved peptide domains.Table 27 shows the SEQ ID NO: of the Pfam domain, the name of the Pfammodel found, the description, the e-value, Pfam score, number ofrepeats, and position of the domain within SEQ ID NO: 558 for theidentified model within the sequence as follows: TABLE 27 SEQ ID Re- NO:Model Description E-value Score peats Position 551 hormone Somatotropin1.6e−57 156.1 1 9-151 hormone family

Using the eMATRIX software package (Stanford University, Stanford,Calif.) (Wu et al., J. Comp. Biol., 6:219-235 (1999), hereinincorporated by reference), the growth hormone-like polypeptide of SEQID NO: 557 was determined to have following the eMATRIX domain hits. Theresults in Table 28 describe: SEQ ID NO of the eMATRIX domain, thecorresponding p-value, subtype, Signature ID number, domain name, theamino acid sequence of the eMATRIX domain and the corresponding positionof the amino acids within SEQ ID NO: 557, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E-Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine: TABLE 28 SEQ Amino acid sequence IDSignature encoded (start and end NO p-value ID NO Domain Name amino acidposition) 560 8.714e−21 BL00266B Somatotropin, prolactinCFSDSIPTSSNMEETQ and related hormones QKSNLELLHISLLLIES proteins RLEPV(79-116) 561 1.923e−14 BL00266A Somatotropin, prolactin LFKEAMLQAHRAHQLand related hormones AIDTYQEFEEAY proteins (35-61) 562 2.862e−11PR00836A SOMATOTROPIN CFSDSIPTSSNMEE HORMONE FAMILY (79-92) SIGNATURE563 4.000e−11 BL00266D Somatotropin, prolactin PGLSLHPEGEGGKWI andrelated hormones NERGREQCP (201-224) proteins 564 7.000e−11 PR00836BSOMATOTROPIN LLHISLLLIESRLEPVR HORMONE FAMILY FL (101-119) SIGNATURE 5653.700e−10 BL00266C Somatotropin, prolactin DDYHLLKDLEEGIQM and relatedhormones LM (135-151) proteins

The growth hormone-like polypeptides, polynucleotides, antibodies andother compositions of the invention are expected to be useful intreating disorders where the growth of limbs and internal organs areeffected, such as dwarfism, giantism, and acromegaly. Growthhormone-like polypeptides, polynucleotides, antibodies and othercompositions of the invention may be used to treat metabolic disorders,including diabetes and obesity. Growth hormone-like polypeptides,polynucleotides, antibodies and other compositions of the invention maybe used to treat inflammation, autoimmune diseases, and to modulateimmune response.

4.7 Neutrophil Gelatinase-Associated Lipocalin-Like (NGALHy)Polypeptides and Polynucleotides

Lipocalins are a diverse family of proteins that are typically small(160-180 residues in length), extracellular proteins that bind smalllipophilic molecules (such as retinol), cell surface receptors, and formcovalent and non-covalent complexes with other soluble macromolecules(reviewed in Flower et al., Biochim. Biophys. Acta 1482:9-24 (2000),herein incorporated by reference). Proteins in the lipocalin familyshare a characteristic conserved lipocalin sequence motif as well as acommon three-dimensional structure forming a β-barrel. Lipocalins havebeen shown to be overexpressed in a variety of diseases including cancerand inflammatory diseases.

Neutrophil gelatinase associated lipocalin (NGAL), a constituent ofneutrophils granules, is a member of the lipocalin family. NGAL ishighly induced in epithelial cells in both inflammatory and neoplasticcolorectal disease (Goetz et al., Biochemistry 39:1935-1941 (2000),herein incorporated by reference). NGAL is proposed to mediateinflammatory responses by sequestering neutrophils chemoattractants,particularly N-formylated tripeptides as well as leukotriene B4 andplatelet activating factor. Lipocalins are mainly extracellular carriersof lipophilic molecules, although exceptions with properties likeprostaglandin synthesis and protease inhibition are observed forspecific lipocalins. Study of lipocalins in cancer has so far beenfocused on the variations in concentration and the modification of theirexpression in distinct cancer forms. In addition, lipocalins have beenassigned a role in cell regulation. Lipocalins have also been usedextensively as biochemical markers of disease (see Xu and Venge,Biochim. Biophys. Acta 1482:298-307 (2000), herein incorporated byreference). The clinical indications relate to almost any field ofmedicine, such as inflammatory disease, cancer, lipid disorders, liverand kidney function.

Two exemplary NGAL-like sequences of the invention (NGALHy1 and NGALHy2)are described below: amino acid sequence SEQ ID NO: 572 (and encodingnucleotide sequence SEQ ID NO: 571), and amino acid SEQ ID NO: 579 (andencoding nucleotide sequence SEQ ID NO: 578).

The NGALHy1 polypeptide of SEQ ID NO: 572 is an approximately 157-aminoacid protein with a predicted molecular mass of approximately 17-kDaunglycosylated. The initial methionine starts at position 192 of SEQ IDNO: 571 and the putative stop codon begins at position 660 of SEQ ID NO:571. A signal peptide of 19 residues is predicted from approximatelyresidue 1 to residue 19 of SEQ ID NO: 572. The extracellular portion isuseful on its own. This can be confirmed by expression in mammaliancells and sequencing of the cleaved product. The signal peptide regionwas predicted using the Neural Network SignalP V1.1 program (Nielsen etal, Int. J. Neural Syst. 8:581-599 (1997)). One of skill in the art willrecognize that the actual cleavage site may be different than thatpredicted by the computer program.

Protein database searches with the BLASTP algorithm (Altschul S. F. etal, J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 572 is homologous to mouse lipocalin (SEQ ID NO: 585) andhuman NGAL precursor (SEQ ID NO: 586).

FIG. 44 shows a multiple sequence alignment of SEQ ID NO: 572 with otherhomologous sequences (SEQ ID NO: 585 and 586) showing conserved regions,wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosineand asterisks (*) indicate identical residues, colons (:) indicateconserved substitutions, and periods (.) indicate distant substitutions.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), NGALHy1 polypeptide of SEQ ID NO: 572 was determined to havefollowing the eMATRIX domain hits. The results describe: correspondingSEQ ID NO: in sequence listing, e-value, subtype, Accession number,domain name, amino acids of the full length protein of SEQ ID NO: 572that correspond to the eMATRIX domain and are displayed in Table 29,wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine:TABLE 29 SEQ ID Accession Domain Amino acid sequence NO: e-value SubtypeNo. Name (start and end position) 576 5.500e−08 13.78 PR00179A LipocalinNQFQGEWFVLGLAGN signature (37-50)

The NGALHy2 polypeptide of SEQ ID NO: 579 is an approximately 200-aminoacid protein with a predicted molecular mass of approximately 22-kDaunglycosylated. The initial methionine starts at position 128 of SEQ IDNO: 578 and the putative stop codon begins at position 725 of SEQ ID NO:578. A signal peptide of 19 residues is predicted from approximatelyresidue 1 to residue 19 of SEQ ID NO: 579. The extracellular portion isuseful on its own. This can be confirmed by expression in mammaliancells and sequencing of the cleaved product. The signal peptide regionwas predicted using the Neural Network SignalP V1.1 program (Nielsen etal, Int. J. Neural Syst. 8:581-599 (1997)). One of skill in the art willrecognize that the actual cleavage site may be different than thatpredicted by the computer program.

Protein database searches with the BLASTP algorithm (Altschul S. F. etal, J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 579 is homologous to mouse lipocalin (SEQ ID NO: 585) andhuman NGAL precursor (SEQ ID NO: 586).

FIG. 44 shows a multiple sequence alignment of SEQ ID NO: 579 widthother homologous sequences (SEQ ID NO: 585 and 586) showing conservedregions, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes and asterisks (*) represent identicalresidues, colons (:) represent conservative substitutions, periods (.)represent semi-conservative substitutions.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), NGALHy2 polypeptide of SEQ ID NO: 579 was determined to havefollowing the eMATRIX domain hits. The results describe: correspondingSEQ ID NO: in sequence listing, e-value, subtype, Accession number,domain name, amino acids of the full length protein of SEQ ID NO: 579that correspond to the eMATRIX domain and are shown in Table 30 below,wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.TABLE 30 SEQ ID Accession Domain Amino acid sequence NO: e-value SubtypeNo. Name (start and end position) 583 5.500e−08 13.78 PR00179A LipocalinNQFQGEWFVLGLAG signature (37-50) 584 7.214e−09 9.56 PR00179B LipocalinVDSDYTQFALMLS signature (121-134)

NGAL forms a heterodimeric complex with matrix metalloproteinase 9(MMP9) which protects MMP9 from degradation and allows MMP9 to degradethe extracellular matrix thereby enhancing tumor cell metastasis (Yan etal., J. Biol. Chem. 276:37258-37265 (2001) herein incorporated byreference). The MMP9/NGAL complex is induced in several cancers and isused as a marker for metastatic cancer. NGAL also modulates the immuneresponse during the acute phase response during inflammation to enhancenon-specific host defenses by binding to and neutralizing pro-infectiousbacterial products, such as the chemoattractant N-formyl-Met-Leu-Phe(Goetz et al., 2000. supra; Logdberg and Wester, Biochim. Biophys. Acta,1482:284-297 (2000), herein incorporated by reference). Circulating NGALlevels are used as a marker for inflammatory conditions, such as cysticfibrosis and acute peritonitis, and are capable of distinguishingbetween bacterial and viral acute infections. NGAL and lipocalins ingeneral, also play a role in cell regulation, cell differentiation, andcell proliferation.

The polypeptides of the invention are expected to have similar functionsas NGAL as a marker for diseases including cancer and inflammatorydiseases, interacting with matrix metalloproteases to modulate cellproliferation, modulation of inflammation by enhancing non-specific hostdefenses, via activities such as binding to bacterial pro-inflammatoryproteins.

The polypeptides, polynucleotides, antibodies and other compositions ofthe invention are expected to be useful in treating the followingdisorders: inflammatory diseases, including bacterial and viralinfections, acute peritonitis, cystic fibrosis, asthma, chronicobstructive pulmonary disease, pulmonary emphysema, Sjogren's syndrome,rheumatoid arthritis; neoplastic colorectal disease, colitis, and otherdisorders in which the barrier of the colorectal mucosa is disrupted;wound healing; cancer, including breast, colorectal, pancreatic,prostate, bladder, renal cancers, colorectal and hepatic tumors,adenocarcinomas, including lung, colon, pancreas; lipid disorders, andmodulating liver and kidney function.

4.8 Mucolipin-Like Polypeptides and Polynucleotides

Mucolipidosis IV (MLIV) is an autosomal recessive neurodegenerativelysosomal storage disorder characterized clinically by psychomotorretardation and ophthalmologic abnormalities including corneal opacitiy,retinal degeneration, and strabismus. Maximal development of the patientis between 12 and 15 months and age of the patients with this diseaseranges from 1 to 40 years. Life expectancy of the patients is not known.Over 80% of the patients diagnosed with MLIV showing severe or mildsymptoms are the Ashknazi Jews. The patients excrete chondroitinsulphate in their urine. The disease is characterized by massiveengorgement of superficial and intermediate epithelial cells of both thecornea and conjunctiva with fine granular material consistent withmucopolysaccharide and concentric lamellar bodies. The storage materialshave been identified as sphingolipids, phospholipids and acidmucopolysaccharides. In this disease, excessive storage of thesematerials is also observed in macrophages, plasma cells, ciliaryepithelial cells, Schwann cells, retinal ganglion cells and vascularendothelial cells.

Unlike other lysosomal storage disorders, MLIV is not associated with alack of lysosomal hydrolases. Instead the MLIV cells display abnormalendocytosis of lipids and accumulate large vesicles indicating that adefect in endocytosis may underlie the disease as shown by Chen, et al.(Chen, et al, Proc. Natl. Acad. Sci. USA. 98:6373-6378 (1998), hereinincorporated by reference). Bassi, et al (Bassi, et al, Human Genet.67:1110-1120, (2000)) also suggested that mucolipin 1 plays an importantrole in endocytosis, a fact that has been borne out by the studies ofFares and Greenwald using C. elegans as an animal model (Fares andGreenwald. Nature Genet. 28:64-68, (2001), herein incorporated byreference). They showed that a loss-of-function mutation in the C.elegans mucolipin 1 homolog, Cup-5 results in increased rate of uptakeof fluid-phase markers, decreased degradation of the endocytosed proteinand and accumulation of large vacuoles. Overexpression of cup-5 causesthe opposite phenotype and rescue with human mucolipin 1 results innormalizing the endocytosis. Cup-5 is also essential for the viabilityand regulates the lysosomes in multiple cell types in C. elegans (Hershet al. Proc Natl Acad Sci USA. 99:4355-4360, (2002), herein incorporatedby reference).

The metabolic defect causing this accumulation has recently beenidentified as dysfunctional endocytosis and the gene responsible hadbeen named mucolipin 1 (Bargal, et al., Nature Genet. 26:20-123, (2000),Bassi, et al., Human Genet. 67:1110-1120, (2000), Sun, et al Hum. Molec.Genet. 9:2471-2478, (2000), all of which are herein incorporated byreference) and it is a transcript of the gene MCOLN1 shown to be locatedon chromosome 19p13.3-p13.2 (Slaugenhaupt et al., Am. J. Hum. Genet.65:773-778, (1999), herein incorporated by reference).

The MLIV gene consists of 14 exons spanning approximately 14 kb ofgenomic DNA and encoding a protein of 580 amino acid in length (Bargal,et al. Nature Genet. 26:120-123, (2000), herein incorporated byreference). The mucolipin protein appears to contain one transmembranehelix in the N-terminal region and at least 5 transmembrane domains ionthe C-terminal half of the protein. This protein localizes on the plasmamembrane and in the C-terminal region shows homology to polycistin-2,the product of the polycystic kidnay disease (PKD2) gene (Bassi, et al.,Human Genet. 67:1110-1120, (2000), herein incorporated by reference).The gene also belongs to a family of transient receptor potentialcalcium ion channels (Sun, et al., Hum. Molec. Genet. 9:2471-2478,(2000), herein incorporated by reference) and may play a role in calciumion transport.

Since the discovery of mucolipin 1 (also known as mucolipidin), at leasttwo other human, three mouse proteins and a C. elegans cup-5 proteinhomologous to the mucolipin 1 have been identified creating a novelfamily of mucolipins. Since, studies on mucolipin 1 and cup-5 have shownthe impact these proteins can have on cell viability, normal cellulartransport, lysosomal storage and resulting in mental retardation,ophthalmic abnormalities such as corneal opacity, retinal degenerationand strabismus, there clearly exists a need for identifying furthermembers of this family of proteins. Identification of such proteins andtheir methods of use to modulate cellular lysosomal transport providetherapeutic compositions and methods of treatments for theabove-mentioned conditions.

The mucolipin-like polypeptide of SEQ ID NO: 588 is an approximately542-amino acid protein with a predicted molecular mass of approximately59.6-kDa unglycosylated. The initial methionine starts at position 1 ofSEQ ID NO: 587 and the putative stop codon begins at position 1629 ofSEQ ID NO: 587: FIG. 45 shows the alignment between the protein in SEQID NO: 588 encoded by SEQ ID NO: 589 and human mucolipin 1 (SEQ ID NO:592), indicating the two sequences share 48% identity over 542 aminoacids wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

The mucolipin-like polypetide is not predicted to have a secretionsignal peptide. The absence of signal peptide region was predicted usingthe Neural Network SignalP V1.1 program (Nielsen et al, Int. J. NeuralSyst. 8:581-599 (1997), herein incorporated by reference). Using theTMpred program, the transmembrane regions of the polypeptide weredetermined. The TMpred program makes a prediction of membrane-spanningregions and their orientation. The algorithm is based on the statisticalanalysis of TMbase, a database of naturally occuring transmembraneproteins. The prediction is made using a combination of severalweight-matrices for scoring. (K. Hofmann & W. Stoffel (1993) TMbase—Adatabase of membrane spanning proteins segments. Biol. Chem.Hoppe-Seyler 374,166, herein incorporated by reference). Onetransmembrane region is predicted be present at the N-terminal end ofthe protein from 35 amino acid to 65 amino acid of SEQ ID NO: 588. Fiveadditional transmembrane regions have been predicted by the Tmpredprogram from amino acid 266 to 282, 324 to 339, amino acid 353 to aminoacid 370, 400 to amino acid 416, and amino acid 466 to amino acid 483 atthe C-terminus of SEQ ID NO: 588.

Protein database searches with the BLASTP algorithm (Altschul, et al.,J. Mol. Evol. 36:290-300 (1993); Altschul et al, J. Mol. Biol. 21:403-10(1990), herein incorporated by reference) indicate that SEQ ID NO: 588is best homologous to mouse mucolipin 2. A multiple sequence alignmentof SEQ ID NO: 588 with other homologous sequences showing conservedregions is shown in FIG. 46.

FIG. 46 shows a multiple sequence alignment between mucolipin-likepolypeptide (SEQ ID NO: 588) and other members of the family: mousemucolipin 2 (SEQ ID NO: 591), human mucolipin 1 (SEQ ID NO: 592), humanmucolipin 3 (SEQ ID NO: 593), and Caenorhabditis elegans CUP-5 (SEQ IDNO: 595). Asterisks (*) indicate that the amino acid at that position isidentical between the different polypetides, colons (:) indicate theamino acids at that postion are conservative replacements and periods(.) indicate the conserved presence of charged amino acids, wherinA=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference), mucolipin-likepolypeptide of SEQ ID NO: 588 revealed its sequence homology to calciumion transport pfam domain. Further description of the Pfam models can befound at http://pfam.wustl.edu/. Pfam domains hits are as follows:calcium ion_transport protein, score=22.4, e-value=0.0001, and aminoacids of the full length protein of SEQ ID NO: 588 that correspond tothe Pfam domain stretching from amino acid 322 to amino acid 482 andnucleotides of the open reading frame of SEQ ID NO: 590 that correspondto the domain.

Mucolipin-like polypeptide contains a conserved serine lipase sitespanning amino acid residues 74 to 90 of SEQ ID NO: 588 that is found inmucolipin 1 and other lipolytic enzymes. FIG. 47 shows an alignment ofthe conserved serine lipase active site between mucolipin-likepolypeptide (SEQ ID NO: 596) and mucolipin 1 (SEQ ID NO: 597) as well asother lipolytic enzymes: H. liph. triacylglycerol lipase hepaticprecursor (SEQ ID NO: 598), H. liph. lipoprotein lipase precursor (SEQID NO: 599) and H. lcat. phosphatidylcholine-sterol acyltransferaseprecursor (SEQ ID NO: 600).

Homologous family members SEQ ID NO: 592 and 595 have the followingactivities: endocytosis, calcium ion transport, apoptosis induction andlipolysis through a conserved serine lipase domain. The polypeptides ofthe invention are expected to have the following activities: based onhomology and analysis of predicted pfam domains, the mucolipin-likepolypeptide is expected to function as not only a calcium ion transportmolecule but also as a serine lipase and play a role in apoptosisinduction, endocytosis and lipid metabolism. The polypeptides,polynucleotides, antibodies and other compositions of the invention areexpected to be useful in treating the following disorders: cholesterolstorage diseases such as MLIV, cardiovascular, ophthalmic and neurologicdiseases as well as diseases associated with apoptosis such asfollicular lymphoma, autoimmune diseases and retinal degeneration.

4.9 Peroxidasin-Like Polypeptides and Polynucleotides

Peroxidasin was first identified and characterized in Drosophila as anovel enzyme-matrix protein based on its hybrid structure which combinesan enzymatically active peroxidase motif with domains that usually occuras parts of interacting extracellular proteins (e.g. cell adhesionmolecules) (Nelson et al, The EMBO Journal; 13:3438-3447(1994),incorporated herein by reference). Peroxidasin is a 1535 amino acidprotein, wherein the amino acid sequence of the peroxidase domain isquite similar to the vertebrate peroxidases myeloperoxidase (MPO),eosinophil peroxidase (EPO), lactoperoxidase (LPO), and thyroidperoxidase (TPO). MPO, EPO, and LPO play key roles in human oxidativedefense (Everse et al, Peroxidases in Chemistry and Biology; (1990),incorporated herein by reference). Since the expression of peroxidasinis accompanied by phagocytosis in the Drosophila embryo, peroxidasin mayalso function in phagocytosis. In addition to its peroxidase domain,peroxidasin possesses six leucine rich repeats (LRR) and fourimmunoglobulin (Ig) repeats. LRR's and Ig loops are involved inprotein-protein interactions and indicate a role for peroxidasin inextracellular matrix consolidation and cell adhesion (Nelson et al, TheEMBO Journal; 13:3438-3447(1994), incorporated herein by reference).

Overexpression of p53, a tumor suppressor protein whose inactivation hasbeen observed in a large number of human cancers, leads to eitherprogrammed cell death (apoptosis) or growth arrest. A human homologue ofDrosophila peroxidasin was shown to be differentially expressed in ahuman colon cancer cell line undergoing p53-dependent apoptosis(Horikoshi et al, Biochem. Biophys. Res. Commun.; 261:864-869(1999),incorporated herein by reference).

Recently, a novel melanoma gene (MG50) was identified which showssignificant similarity to peroxidasin (Mitchell et al, Cancer Research;60:6448-6456(2000), incorporated herein by reference). There is evidencethat suggests MG50 is relatively restricted to tumors such as melanoma,breast cancer, ovarian cancer, and glioblastoma. In contrast, MG50appears to be absent from archived specimens of normal tissues, with theexception of skin (Mitchell et al, Cancer Research; 60:6448-6456(2000),incorporated herein by reference). Since MG50 seems to be relativelytumor associated, it was hypothesized that MG50 could be a potentiallyuseful immunogen and target for immunotherapy.

There exists a need for identifying further members of this family ofproteins.

Six exemplary peroxidasin-like sequences of the invention are disclosedbelow: amino acid sequence SEQ ID NO: 602 (and encoding nucleotidesequence SEQ ID NO: 601), amino acid sequence SEQ ID NO: 618 (andencoding nucleotide sequence SEQ ID NO: 617), amino acid sequence SEQ IDNO: 622 (and encoding nucleotide sequence SEQ ID NO: 621), amino acidsequence SEQ ID NO: 626 (and encoding nucleotide sequence SEQ ID NO:625), amino acid sequence SEQ ID NO: 607 (and encoding nucleotidesequence SEQ ID NO: 606), amino acid sequence SEQ ID NO: 612 (andencoding nucleotide sequence SEQ ID NO: 611).

The peroxidasin-like polypeptide of SEQ ID NO: 603 is an approximately1507-amino acid protein with a predicted molecular mass of approximately166 kDa unglycosylated. The initial methionine starts at position 261 ofSEQ ID NO: 601 and the putative stop codon begins at position 4782 ofSEQ ID NO: 601. A signal peptide of twenty three residues (SEQ ID NO:604) is predicted from approximately residue 1 to residue 23 of SEQ IDNO: 602. The extracellular portion is useful on its own. This can beconfirmed by expression in mammalian cells and sequencing of the cleavedproduct. The signal peptide region was predicted using the NeuralNetwork SignalP V1.1 program (Nielsen et al, Int. J. Neural Syst.8:581-599 (1997)). One of skill in the art will recognize that theactual cleavage site may be different than that predicted by thecomputer program.

Using the TMpred program (Hofmann and Stoffel, Biol. Chem. 374:166(1993), herein incorporated by reference), SEQ ID NO: 602 is predictedto have transmembrane domains at approximately residue 5 to residue 26,residue 505 to residue 518, residue 593 to residue 608, and residue 1086to residue 1104. Removal of one or more transmembrane domains rendersfragments that can be useful on their own. One example is a fragmentfrom residue 24 to residue 504 of SEQ ID NO: 602. One of skill in theart will recognize that the actual transmembrane domains may bedifferent than that predicted by the computer program.

Protein database searches with the BLASTP algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 602 is homologous to peroxidasin-like proteins.

FIG. 48 shows the BLASTP amino acid sequence alignment betweenperoxidasin-like polypeptide SEQ ID NO: 602 and human peroxidasin-likeprotein (also known as melanoma-associated antigen, MG50) (SEQ ID NO:616), indicating that the two sequences share: 73% similarity and 60%identity over 855 amino acid residues, 73% similarity and 57% identityover a distinct 464 amino acid residues, and 75% similarity and 60%identity over a distinct 86 amino acid residues, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference) SEQ ID NO: 602 wasexamined for domains with homology to known conserved peptide domains.Table 31 shows the name of the Pfam model found, the description, thee-value, Pfam score, number of repeats, and position of the domain(s)within SEQ ID NO: 602 for the identified model within the sequence asfollows: TABLE 31 Re- Model Description E-value Score peats Positionperox- Peroxidase 1.1e−40 148.6 1  770-1208 idase Ig Immunoglobulin4.1e−35 118.2 4 224-283 domain 320-376 416-472 533-590 LRR Leucine RichRepeat 1.4e−19 78.5 5 51-74 75-98  99-122 123-146 147-171 LRRCT Leucinerich repeat 9.1e−11 49.2 1 156-208 C-terminal domain vwc von Willebrandfactor   7e−08 39.6 1 1439-1494 type C domain TILa TILa domain 0.02312.0 1 1438-1491

Using the eMATRIX software package (Stanford University, Stanford,Calif.) (Wu et al., J. Comp. Biol., 6:219-235 (1999), hereinincorporated by reference), the peroxidasin-like polypeptide of SEQ IDNO: 602 was determined to have following the eMATRIX domain hits. Theresults in Table 32 describe: the eMATRIX domain name, the correspondingp-value, Signature ID number, and the corresponding position of thedomain within SEQ ID NO: 603: TABLE 32 Signature Name p-value ID NOPosition ANIMAL HAEM 3.118e−22 PR00457E 1041-1067 PEROXIDASE SIGNATUREANIMAL HAEM 4.194e−21 PR00457D 1016-1036 PEROXIDASE SIGNATURE ANIMALHAEM 1.675e−13 PR00457C  998-1016 PEROXIDASE SIGNATURE ANIMAL HAEM5.680e−13 PR00457H 1292-1306 PEROXIDASE SIGNATURE ANIMAL HAEM 4.750e−12PR00457F 1094-1104 PEROXIDASE SIGNATURE ANIMAL HAEM 8.615e−12 PR00457G1221-1241 PEROXIDASE SIGNATURE VWFC domain 3.250e−10 BL01208B 1480-1494proteins ANIMAL HAEM 3.411e−10 PR00457B 846-861 PEROXIDASE SIGNATUREReceptor tyrosine 1.000e−09 BL00240B 325-348 kinase class III proteinsRECEPTOR FC 4.581e−09 PD01270A 304-343 IMMUNOGLOBULIN AFFIN.LEUCINE-RICH 7.480e−09 PR00019B 73-86 REPEAT SIGNATURE

A first variant of SEQ ID NO: 602 is SEQ ID NO: 618. The variant is anapproximately 1538 amino acid protein with a predicted molecular mass ofapproximately 169 kDa unglycosylated. The initial methionine starts atposition 12 of SEQ ID NO: 617, and the putative stop codon begins atposition 4626 of SEQ ID NO: 617. A signal peptide of 54 residues ispredicted from approximately residue 1 to residue 54 of SEQ ID NO: 618.The extracellular portion is useful on its own. This can be confirmed byexpression in mammalian cells and sequencing of the cleaved product. SEQID NO: 618 differs from SEQ ID NO: 602 at the N-terminus where itcontains an additional 31 amino acids. The remainder of SEQ ID NO: 618is identical to SEQ ID NO: 602. Therefore, SEQ ID NO: 618 comprises SEQID NO: 602. The signal peptide region was predicted using the NeuralNetwork SignalP V1.1 program (Nielsen et al, Int. J. Neural Syst.8:581-599 (1997)). One of skill in the art will recognize that theactual cleavage site may be different than that predicted by thecomputer program.

Using the TMpred program (Hofmann and Stoffel, Biol. Chem. 374:166(1993), herein incorporated by reference), SEQ ID NO: 618 is predictedto have a transmembrane domain at approximately residue 525 to residue550. Removal of the transmembrane domain renders fragments that can beuseful on their own. One of skill in the art will recognize that theactual transmembrane domain may be different than that predicted by thecomputer program.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference) SEQ ID NO: 618 wasexamined for domains with homology to known conserved peptide domains.Table 33 shows the name of the Pfam model found, the description, thee-value, Pfam score, number of repeats, and position of the domain(s)within SEQ ID NO: 618 for the identified model within the sequence asfollows: TABLE 33 Re- Model Description E-value Score peats PositionAn_per- Animal haem   1e−192 653.6 1  801-1340  oxidase peroxidase igImmunoglobulin 1.4e−32 121.6 4 255-314: domain 351-407: 447-503:564-621  LRR Leucine Rich 3.3e−16 63.7 5  82-105: Repeat 106-129:130-153: 154-177: 178-189  LRRCT Leucine rich 1.2e−14 47.5 1 187-239 repeat C- terminal domain vwc von Willebrand 1.2e−09 38.0 1 1470-1525 factor type C domain TILa TILa domain 0.0017 16.9 1 1469-1508  LRRNTLeucine rich 0.025 14.9 1 54-80  repeat N- terminal domain

Using the eMATRIX software package (Stanford University, Stanford,Calif.) (Wu et al., J. Comp. Biol., 6:219-235 (1999), hereinincorporated by reference), the peroxidasin-like polypeptide of SEQ IDNO: 618 was determined to have following the eMATRIX domain hits. Theresults in Table 34 describe: the eMATRIX domain name, the correspondingp-value, Signature ID number, and the corresponding position of thedomain within SEQ ID NO: 618: TABLE 34 Signature Name p-value ID NOPosition ANIMAL HAEM 8.45e−24 PR00457E 1072-1098 PEROXIDASE SIGNATURE VANIMAL HAEM 1.53e−20 PR00457D 1047-1067 PEROXIDASE SIGNATURE IV ANIMALHAEM 9.42e−15 PR00457C 1029-1047 PEROXIDASE SIGNATURE III ANIMAL HAEM4.48e−14 PR00457G 1252-1272 PEROXIDASE SIGNATURE VII ANIMAL HAEM5.85e−13 PR00457H 1323-1337 PEROXIDASE SIGNATURE VIII ANIMAL HAEM6.32e−12 PR00457F 1125-1135 PEROXIDASE SIGNATURE VI LEUCINE RICH1.00e−10 IPB000483 187-201 REPEAT C- TERMINAL DOMAIN ANIMAL HAEM2.29e−10 PR00457B 877-892 PEROXIDASE SIGNATURE II IMMUNOGLOBULIN2.80e−10 IPB003006B 383-420 AND MAJOR HISTO- COMPATIBILITY COMPLEXDOMAIN IMMUNOGLOBULIN 8.92e−10 IPB003006B 479-516 AND MAJOR HISTO-COMPATIBILITY COMPLEX DOMAIN IMMUNOGLOBULIN 9.28e−10 IPB003006B 290-327AND MAJOR HISTO- COMPATIBILITY COMPLEX DOMAIN

A second variant of SEQ ID NO: 602 is SEQ ID NO: 622. The splice siteoccurs after nucleotide 329 of SEQ ID NO: 601. The variant is anapproximately 1400 amino acid protein with a predicted molecular mass ofapproximately 154 kDa unglycosylated. The initial methionine starts atposition 103 of SEQ ID NO: 621, and the putative stop codon begins atposition 4303 of SEQ ID NO: 621. A signal peptide of 23 residues ispredicted from approximately residue 1 to residue 23 of SEQ ID NO: 622.The extracellular portion is useful on its own. This can be confirmed byexpression in mammalian cells and sequencing of the cleaved product. Thesignal peptide region was predicted using the Neural Network SignalPV1.1 program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)). Oneof skill in the art will recognize that the actual cleavage site may bedifferent than that predicted by the computer program.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference) SEQ ID NO: 622 wasexamined for domains with homology to known conserved peptide domains.Table 35 shows the name of the Pfam model found, the description, thee-value, Pfam score, number of repeats, and position of the domain(s)within SEQ ID NO: 622 for the identified model within the sequence asfollows: TABLE 35 Re- Model Description E-value Score peats PositionAn_per- Animal haem   1e−192 653.6 1  663-1202 oxidase peroxidase IgImmunoglobulin 7.8e−25 95.7 4 201-260 domain 297-353 393-449 514-532 LRRLeucine Rich 2.7e−14 57.0 4 51-74 Repeat 75-98  99-122 123-146 Vwc vonWillebrand 1.2e−09 38.0 1 1332-1387 factor type C domain TILa TILadomain 0.0017 16.9 1 1331-1370 LRRNT Leucine rich 0.025 14.9 1 23-49repeat N- terminal domain

Using the eMATRIX software package (Stanford University, Stanford,Calif.) (Wu et al., J. Comp. Biol., 6:219-235 (1999), hereinincorporated by reference), the peroxidasin-like polypeptide of SEQ IDNO: 622 was determined to have following the eMATRIX domain hits. Theresults in Table 36 describe: the eMATRIX domain name, the correspondingp-value, Signature ID number, and the corresponding position of thedomain within SEQ ID NO: 622: TABLE 36 Signature Name p-value ID NOPosition ANIMAL HAEM 8.45e−24 PR00457E 934-960 PEROXIDASE SIGNATURE VANIMAL HAEM 1.53e−20 PR00457D 909-929 PEROXIDASE SIGNATURE IV ANIMALHAEM 9.42e−15 PR00457C 891-909 PEROXIDASE SIGNATURE III ANIMAL HAEM4.48e−14 PR00457G 1114-1134 PEROXIDASE SIGNATURE VII ANIMAL HAEM5.85e−13 PR00457H 1185-1199 PEROXIDASE SIGNATURE VIII ANIMAL HAEM6.32e−12 PR00457F 987-997 PEROXIDASE SIGNATURE VI ANIMAL HAEM 2.29e−10PR00457B 739-754 PEROXIDASE SIGNATURE II IMMUNOGLOBULIN 2.80e−10IPB003006B 329-366 AND MAJOR HISTOCOMPATIBILITY COMPLEX DOMAINIMMUNOGLOBULIN 8.92e−10 IPB003006B 425-462 AND MAJOR HISTOCOMPATIBILITYCOMPLEX DOMAIN IMMUNOGLOBULIN 9.28e−10 IPB003006B 236-273 AND MAJORHISTOCOMPATIBILITY COMPLEX DOMAIN LEUCINE-RICH 6.73e−09 PR00019B 73-86REPEAT SIGNATURE II

A third variant of SEQ ID NO: 602 is SEQ ID NO: 626. The splice siteoccurs after nucleotide 329 of SEQ ID NO: 601. The variant is anapproximately 1439 amino acid protein with a predicted molecular mass ofapproximately 158 kDa unglycosylated. The initial methionine starts atposition 261 of SEQ ID NO: 625, and the putative stop codon begins atposition 4578 of SEQ ID NO: 625. A signal peptide of 23 residues ispredicted from approximately residue 1 to residue 23 of SEQ ID NO: 626.The extracellular portion is useful on its own. This can be confirmed byexpression in mammalian cells and sequencing of the cleaved product. Thesignal peptide region was predicted using the Neural Network SignalPV1.1 program (Nielsen et al, Int. J. Neural Syst. 8:581-599 (1997)). Oneof skill in the art will recognize that the actual cleavage site may bedifferent than that predicted by the computer program.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference) SEQ ID NO: 626 wasexamined for domains with homology to known conserved peptide domains.Table 37 shows the name of the Pfam model found, the description, thee-value, Pfam score, number of repeats, and position of the domain(s)within SEQ ID NO: 626 for the identified model within the sequence asfollows: TABLE 37 Re- Model Description E-value Score peats PositionAn_per- Animal haem 9.1e−194 657.1 1  702-1241 oxidase peroxidase igImmunoglobulin 6.2e−34 126.2 4 224-283 domain 320-376 416-466 501-558LRR Leucine Rich 3.3e−16 63.7 5 51-74 Repeat 75-98  99-122 123-146147-158 LRRCT Leucine rich 1.2e−14 47.5 1 156-208 repeat C- terminaldomain vwc von Willebrand 1.2e−09 38.0 1 1371-1426 factor type C domainTILa TILa domain 0.0017 16.9 1 1370-1409 LRRNT Leucine rich 0.025 14.9 123-49 repeat N- terminal domain

Using the eMATRIX software package (Stanford University, Stanford,Calif.) (Wu et al., J. Comp. Biol., 6:219-235 (1999), hereinincorporated by reference), the peroxidasin-like polypeptide of SEQ IDNO: 626 was determined to have following the eMATRIX domain hits. Theresults in Table 38 describe: the eMATRIX domain name, the correspondingp-value, Signature ID number, and the corresponding position of thedomain within SEQ ID NO: 626: TABLE 38 Signature Name p-value ID NOPosition ANIMAL HAEM 8.45e−24 PR00457E 973-999 PEROXIDASE SIGNATURE VANIMAL HAEM 1.53e−20 PR00457D 948-968 PEROXIDASE SIGNATURE IV ANIMALHAEM 9.42e−15 PR00457C 930-948 PEROXIDASE SIGNATURE III ANIMAL HAEM4.48e−14 PR00457G 1153-1173 PEROXIDASE SIGNATURE VII ANIMAL HAEM5.85e−13 PR00457H 1224-1238 PEROXIDASE SIGNATURE VIII ANIMAL HAEM6.32e−12 PR00457F 1026-1036 PEROXIDASE SIGNATURE VI LEUCINE RICH1.00e−10 IPB000483 156-170 REPEAT C- TERMINAL DOMAIN ANIMAL HAEM2.29e−10 PR00457B 778-793 PEROXIDASE SIGNATURE II IMMUNOGLOBULIN2.80e−10 IPB003006B 352-389 AND MAJOR HISTOCOMPATIBILITY COMPLEX DOMAINIMMUNOGLOBULIN 8.92e−10 IPB003006B 442-479 AND MAJOR HISTOCOMPATIBILITYCOMPLEX DOMAIN IMMUNOGLOBULIN 9.28e−10 IPB003006B 259-296 AND MAJORHISTOCOMPATIBILITY COMPLEX DOMAIN

Protein database searches with the BLASTP algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatthe variant sequences SEQ ID NO: 619, 623 and 627 are homologous to thehuman peroxidasin-like protein (accession number BAA13219.1) that isalso known as the melanoma-associated antigen MG50 (Accession numberAF200349_(—)1) (SEQ ID NO: 617).

FIG. 49 shows a multiple sequence alignment between the three variantsof peroxidase-like polypeptide SEQ ID NO: 602, namely SEQ ID NO: 618,624, and 626, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes, asterisks (*) represent identicalresidues, colons (:) represent conservative substitutions, and periods(.) represent semi-conservative substitutions.

The peroxidasin-like polypeptide of SEQ ID NO: 607 is an approximately1463-amino acid protein with a predicted molecular mass of approximately161 kDa unglycosylated. The initial methionine starts at position 145 ofSEQ ID NO: 606 and the putative stop codon begins at position 4534 ofSEQ ID NO: 606. A signal peptide of twenty three residues (SEQ ID NO:609) is predicted from approximately residue 1 to residue 23 of SEQ IDNO: 607. The extracellular portion is useful on its own. This can beconfirmed by expression in mammalian cells and sequencing of the cleavedproduct. The signal peptide region was predicted using the NeuralNetwork SignalP V1.1 program (Nielsen et al, Int. J. Neural Syst.8:581-599 (1997)). One of skill in the art will recognize that theactual cleavage site may be different than that predicted by thecomputer program.

Using the TMpred program (Hofmann and Stoffel, Biol. Chem. 374:166(1993), herein incorporated by reference), SEQ ID NO: 607 is predictedto have transmembrane domains at approximately residue 6 to residue 20,residue 585 to residue 600, and residue 1042 to residue 1060. Removal ofone or more transmembrane domains renders fragments that can be usefulon their own. One example is a fragment from residue 24 to residue 584of SEQ ID NO: 607. One of skill in the art will recognize that theactual transmembrane domains may be different than that predicted by thecomputer program.

Protein database searches with the BLASTP algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 608 is homologous to peroxidasin-like proteins.

FIG. 50 shows the BLASTP amino acid sequence alignment betweenperoxidasin-like polypeptide SEQ ID NO: 607 and human peroxidasin-likeprotein (also known as melanoma-associated antigen, MG50) (SEQ ID NO:616), indicating that the two sequences share 74% similarity and 60%identity over 1459 amino acid residues, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference) SEQ ID NO: 607 wasexamined for domains with homology to known conserved peptide domains.Table 39 shows the name of the Pfam model found, the description, thee-value, Pfam score, number of repeats, and position of the domain(s)within SEQ ID NO: 607 for the identified model within the sequence asfollows: TABLE 39 Re- Model Description E-value Score peats Positionperox- Peroxidase 1.1e−40 148.6 1  726-1164 idase Ig Immunoglobulin6.2e−36 120.8 4 248-307 domain 344-400 440-490 525-582 LRR Leucine RichRepeat 2.3e−22 87.7 6 51-74 75-98  99-122 123-146 147-170 171-195 LRRCTLeucine rich repeat 9.1e−11 49.2 1 180-232 C-terminal domain vwc vonWillebrand factor   7e−08 39.6 1 1395-1450 type C domain TILa TILadomain 0.023 12.0 1 1394-1447

Using the eMATRIX software package (Stanford University, Stanford,Calif.) (Wu et al., J. Comp. Biol., 6:219-235 (1999), hereinincorporated by reference), the peroxidasin-like polypeptide of SEQ IDNO: 607 was determined to have following the eMATRIX domain hits. Theresults in Table 40 describe: the eMATRIX domain name, the correspondingp-value, Signature ID number, and the corresponding position of thedomain within SEQ ID NO: 607: TABLE 40 Signature Name p-value ID NOPosition ANIMAL HAEM 3.118e−22 PR00457E 973-999 PEROXIDASE SIGNATUREANIMAL HAEM 4.194e−21 PR00457D 948-968 PEROXIDASE SIGNATURE ANIMAL HAEM1.675e−13 PR00457C 930-948 PEROXIDASE SIGNATURE ANIMAL HAEM 5.680e−13PR00457H 1224-1238 PEROXIDASE SIGNATURE ANIMAL HAEM 4.750e−12 PR00457F1026-1036 PEROXIDASE SIGNATURE ANIMAL HAEM 8.615e−12 PR00457G 1153-1173PEROXIDASE SIGNATURE VWFC domain 3.250e−10 BL01208B 1412-1426 proteinsANIMAL HAEM 3.411e−10 PR00457B 778-793 PEROXIDASE SIGNATURE Receptortyrosine 1.000e−09 BL00240B 325-348 kinase class III proteinsLEUCINE-RICH 7.480e−09 PR00019B 73-86 REPEAT SIGNATURE RECEPTOR FC7.677e−09 PD01270A 304-343 IMMUNOGLOBULIN AFFIN.

The peroxidasin-like polypeptide of SEQ ID NO: 612 is an approximately1439-amino acid protein with a predicted molecular mass of approximately158 kDa unglycosylated. The initial methionine starts at position 145 ofSEQ ID NO: 611 and the putative stop codon begins at position 4462 ofSEQ ID NO: 611. A signal peptide of twenty-three residues (SEQ ID NO:614) is predicted from approximately residue 1 to residue 23 of SEQ IDNO: 612. The extracellular portion is useful on its own. This can beconfirmed by expression in mammalian cells and sequencing of the cleavedproduct. The signal peptide region was predicted using the NeuralNetwork SignalP V1.1 program (Nielsen et al, Int. J. Neural Syst.8:581-599 (1997)). One of skill in the art will recognize that theactual cleavage site may be different than that predicted by thecomputer program.

Using the TMpred program (Hofmann and Stoffel, Biol. Chem. 374:166(1993), herein incorporated by reference), SEQ ID NO: 612 is predictedto have transmembrane domains at approximately residue 6 to residue 20,residue 561 to residue 576, and residue 1018 to residue 1036. Removal ofone or more transmembrane domains renders fragments that can be usefulon their own. One example is a fragment from residue 24 to residue 560of SEQ ID NO: 612. One of skill in the art will recognize that theactual transmembrane domains may be different than that predicted by thecomputer program.

Protein database searches with the BLASTP algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 612 is homologous to peroxidasin-like proteins.

FIG. 51 shows the BLASTP amino acid sequence alignment betweenperoxidasin-like polypeptide SEQ ID NO: 612 and human peroxidasin-likeprotein (melanoma-associated antigen, MG50) (SEQ ID NO: 616), indicatingthat the two sequences share: 74% similarity and 60% identity over 1386amino acid residues, and 69% similarity and 47% identity over a distinct155 amino acid residues, wherein A=Alanine, C=Cysteine, D=Aspartic Acid,E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine,K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference) SEQ ID NO: 612 wasexamined for domains with homology to known conserved peptide domains.Table 41 shows the name of the Pfam model found, the description, thee-value, Pfam score, number of repeats, and position of the domain(s)within SEQ ID NO: 612 for the identified model within the sequence asfollows: TABLE 41 Re- Model Description E-value Score peats Positionperox- Peroxidase 1.1e−40 148.6 1  702-1140 idase Ig Immunoglobulin6.2e−36 120.8 4 224-283 domain 320-376 416-466 501-558 LRR Leucine RichRepeat 1.2e−18 75.4 5 51-74 75-98  99-122 123-146 147-171 LRRCT Leucinerich repeat 9.1e−11 49.2 1 156-208 C-terminal domain vwc von Willebrandfactor   7e−08 39.6 1 1371-1426 type C domain TILa TILa domain 0.02312.0 1 1370-1423

Using the eMATRIX software package (Stanford University, Stanford,Calif.) (Wu et al., J. Comp. Biol., 6:219-235 (1999), hereinincorporated by reference), the peroxidasin-like polypeptide of SEQ IDNO: 612 was determined to have following the eMATRIX domain hits. Theresults in Table 42 describe: the eMATRIX domain name, the correspondingp-value, Signature ID number, and the corresponding position of thedomain within SEQ ID NO: 613: TABLE 42 Signature Name p-value ID NOPosition ANIMAL HAEM 3.118e−22 PR00457E 973-999 PEROXIDASE SIGNATUREANIMAL HAEM 4.194e−21 PR00457D 948-968 PEROXIDASE SIGNATURE ANIMAL HAEM1.675e−13 PR00457C 930-948 PEROXIDASE SIGNATURE ANIMAL HAEM 5.680e−13PR00457H 1224-1238 PEROXIDASE SIGNATURE ANIMAL HAEM 4.750e−12 PR00457F1026-1036 PEROXIDASE SIGNATURE ANIMAL HAEM 8.615e−12 PR00457G 1153-1173PEROXIDASE SIGNATURE VWFC domain 3.250e−10 BL01208B 1412-1426 proteinsANIMAL HAEM 3.411e−10 PR00457B 778-793 PEROXIDASE SIGNATURE Receptortyrosine 1.000e−09 BL00240B 325-348 kinase class III proteinsLEUCINE-RICH 7.480e−09 PR00019B 73-86 REPEAT SIGNATURE RECEPTOR FC7.677e−09 PD01270A 304-343 IMMUNOGLOBULIN AFFIN.

Peroxidasin-like polypeptides are expected to play roles in phagocytosisand cell adhesion and possess peroxidase-like enzymatic activity.Additionally, peroxidasin-like polypeptides may serve as tumor markersand tumor-specific antigens for immunotherapy.

Immunotherapy provides a method of harnessing the immune system to treatvarious pathological states, including cancer, autoimmune disease,transplant rejection, hyperproliferative conditions, and allergicreactions.

Antibody therapy for cancer involves the use of antibodies, or antibodyfragments, against a tumor antigen to target antigen-expressing cells.Antibodies, or antibody fragments, may have direct or indirect cytotoxiceffects or may be conjugated or fused to cytotoxic moieties. Directeffects include the induction of apoptosis, the blocking of growthfactor receptors, and anti-idiotype antibody formation. Indirect effectsinclude antibody-dependent cell-mediated cytotoxicity (ADCC) andcomplement-mediated cellular cytotoxicity (CMCC). When conjugated orfused to cytotoxic moieties, the antibodies, or fragments thereof,provide a method of targeting the cytotoxicity towards the tumor antigenexpressing cells. (Green, et al., Cancer Treatment Reviews, 26:269-286(2000), incorporated herein by reference).

For example, Rituximab (Rituxan®) is a chimeric antibody directedagainst CD20, a B cell-specific surface molecule found on >95% of B-cellnon-Hodgkin's lymphoma (Press, et al., Blood 69:584-591 (1987),incorporated herein by reference; Malony, et al., Blood 90:2188-2195(1997), incorporated herein by reference). Rituximab induces ADCC andinhibits cell proliferation through apoptosis in malignant B cells invitro (Maloney, et al., Blood 88:637a (1996), incorporated herein byreference). Rituximab is currently used as a therapy for advanced stageor relapsed low-grade non-Hodgkin's lymphoma, which has not responded toconventional therapy.

Active immunotherapy, whereby the host is induced to initiate an immuneresponse against its own tumor cells can be achieved using therapeuticvaccines. One type of tumor-specific vaccine uses purified idiotypeprotein isolated from tumor cells, coupled to keyhole limpet hemocyanin(KLH) and mixed with adjuvant for injection into patients with low-gradefollicular lymphoma (Hsu, et al., Blood 89:3129-3135 (1997),incorporated herein by reference). Another type of vaccine usesantigen-presenting cells (APCs), which present antigen to naïve T cellsduring the recognition and effector phases of the immune response.Dendritic cells, one type of APC, can be used in a cellular vaccine inwhich the dendritic cells are isolated from the patient, co-culturedwith tumor antigen and then reinfused as a cellular vaccine (Hsu, etal., Nat. Med. 2:52-58 (1996), incorporated herein by reference). Immuneresponses can also be induced by injection of naked DNA. Plasmid DNAthat expresses bicistronic mRNA encoding both the light and heavy chainsof tumor idiotype proteins, such as those from B cell lymphoma, wheninjected into mice, are able to generate a protective, anti-tumorresponse (Singh, et al., Vaccine 20:1400-1411 (2002), incorporatedherein by reference).

The peroxidasin-like polypeptides, polynucleotides, antibodies and othercompositions of the invention are expected to be useful in providingtherapeutic compositions and diagnostic methods for treating andidentifying cancer, hyperproliferative disorders, auto-immune diseases,and organ transplant rejection.

4.10 Synaptic Associated 90/Postsynaptic Density Protein 95kDa-Associated Protein-Like Polypeptides and Polynucleotides

Synaptic associated protein 90/postsynaptic density protein 95kDa-associated proteins (SAPAPs) (Takeuchi et al., J. Biol. Chem.272:11943-11951 (1997), herein incorporated by reference), also calledGKAPs (Guanylate kinase-associated proteins) (Kim et al., J Cell Biol.136:669-678 (1997) (Naisbitt et al., J Neurosci. 17:5687-5696 (1997),both herein incorporated by reference) or DAPs (hDLG-associatedproteins) (Satoh et al., Genes Cells. 2:415-424 (1997), hereinincorporated by reference), are major molecular constituents ofpostsynaptic densities. Pre- and postsynaptic specializations are formedgradually during brain development and in the adult nervous systemcontribute to regulate synaptic transmission. (Kawashima et al., FEBSLett. 418:301-304 (1997), herein incorporated by reference). SAPAPs areassociated with the postsynaptic density protein 95 kDa/synapticassociated protein 90 (PSD-95/SAP90) which belongs to the large familyof synaptic membrane-associated guanylate kinases (MAGUKs). This classof proteins contains characteristic domains, which mediateprotein/protein interactions, including PDZ, SH3, and guanylate kinasedomains. These domains enable the MAGUKs to build scaffolds of synapticcomponents that include: a) ion channels and neurotransmitter receptorsvia their NH2-terminal PDZ domains (for example NMDA receptors andpotassium channels) (Kim et al., J. Cell Biol. 136:669-678 (1997),herein incorporated by reference); b) intracellular signaling molecules;and c) cytoskeletal proteins (Naisbitt et al., J Neurosci. 17:5687-5696(1997), herein incorporated by reference). Thus PSD-95 family proteinsfunction as molecular anchors for coupling synaptic receptors and ionchannels to downstream signaling molecules and cytoskeleton. Thehypothesis that SAPAPs play a role in the molecular organization ofsynapses and neuronal cell signaling is suggested by the followingobservations: SAPAPs bind directly to a) the guanylate kinase domain ofthe postsynaptic density protein 95 (PSD-95) family, b) members of thedynein light chain family (Naisbitt et al., J Neurosci. 20:45244534(2000), herein incorporated by reference), which are implicated insynaptic remodeling, and c) Shank, which is a protein that linksdifferent glutamate receptor complexes (NMDA and metabotropic) (Sangmiet al., J. Biol. Chem. 274:29510-29518 (1999), herein incorporated byreference). Thus SAPAPs may orchestrate functional interactions betweenmetabotropic and ionotropic systems. This is relevant in the context ofsynaptic transmission and stabilization since SAPAPs also modulate NMDAchannel conductance (Yamada et al., FEBS Lett. 458:295-298 (1999),herein incorporated by reference), interact with neuronal nitric oxidesynthase (Haraguchi et al., Genes Cells. 5:905-911 (2000), hereinincorporated by reference), neurofilaments (Hirao et al., Genes Cells.5:203-210 (2000), herein incorporated by reference), and synapticscaffolding molecule (S-SCAM; Hirao et al., J. Biol. Chem. 275:2966-2972(2000), herein incorporated by reference). Thus, SAPAPs may be involvedin the molecular organization of synapses and neuronal cell signaling.

Clones of the SAPAP family have been isolated (Boeckers et al., BiochemBiophys Res Commun. 264:247-252 (1999), herein incorporated byreference). SAP proteins are expressed not only in the synapse, but alsoin epithelial cells (Fujita and Kurachi, Biochem Biophys Res Commun.269:1-6 (2000), herein incorporated by reference). Taken together, it isstrongly suggested that various SAPAP proteins help SAPs performspecific functions in different tissues. Therefore, it is important toidentify other members of this family of proteins.

The SAPAP-like polypeptide of SEQ ID NO: 630 is an approximately979-amino acid protein with a predicted molecular mass of approximately107.7-kDa unglycosylated. The initial methionine starts at position 1 ofSEQ ID NO: 629 and the putative stop codon begins at position 2938 ofSEQ ID NO: 629.

Protein database searches with the BLASTP algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 630 is homologous to rat SAPAP (gi|17374684).

FIG. 52 shows a BLASTP amino acid sequence alignment between SAPAP-likepolypeptide (SEQ ID NO: 630) and rat SAPAP3 (SEQ ID NO: 633), indicatingthat the two sequences share 96% similarity over amino acids 1-979 ofSEQ ID NO: 630 and 95% identity over the same amino acids 1-979 of SEQID NO: 630, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res.,26:320-322 (1998) herein incorporated by reference), SAPAP-likepolypeptide of SEQ ID NO: 630 revealed its structural homology toGuanylate-kinase-associated protein (GKAP) corresponding to amino acidsof 621-979 of the full length protein of SEQ ID NO: 630 that correspondto the Pfam domain and nucleotides of 1858-2937 the open reading frameof SEQ ID NO: 631 and is shown in Table 43. Further description of thePfam models can be found at http://pfam.wustl.edu/. TABLE 43 SEQ Aminoacid sequence ID (start and NO: Domain E-value Score end position) 632Guanylate- 7e−292 983.7 ELRSLARQRKWRPSIGVQVET kinase-ISDSDTENRSRREFHSIGVQV associated EEDKRRARFKRSNSVTAGVQA proteinDLELEGLAGLATVATEDKALQ FGRSFQRHASEPQPGPRAPTY SVFRTVHTQGQWAYREGYPLPYEPPATDGSPGPAPAPTPCPG AGRRDSWIERGSRSLPDSGRA SPCPRDGEWFIKMLRAEVEKLEHWCQQMEREAEDYELPEEIL EKIRSAVGSTQLLLSQKVQQF FRLCQQSMDPTAFPVPTFQDLAGFWDLLQLSIEDVTLKFLEL QQLKANSWKLLEPKEEKKVPP PIPKKPLRGRGVPVKERSLDSVDRQRQEARKRLLAAKRAASF RHSSATESADSIEIYIPEAQT RL (621-979)

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al, J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference in its entirety), SAPAP-like polypeptide of SEQ ID NO: 630 wasdetermined to have following eMATRIX domain hits. The results in Table44 describe: corresponding SEQ ID NO: in sequence listing, e-value,subtype, Accession number, name, position of the domain in thefull-length protein, and the amino acid sequence, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F—Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. TABLE 44 SEQ Amino Acid ID AccessionSequence (start NO: e-value subtype No. Name and end position) 6345.97e−11 5.92 PR01256B Otx 1 transcription TSHHHHHHHH factor signatureII HHH (221-233) 635 7.51e−11 5.92 PR01256B Otx 1 transcriptionGPHTSHHHHH factor signature II HHH (218-230) 636 2.35e−10 5.92 PR01256BOtx 1 transcription PHTSHHHHHH factor signature II HHH (219-231) 6372.11e−09 5.92 PR01256B Otx 1 transcription HTSHHHHHHH factor signatureII HHH (220-232) 638 2.31−e09 5.92 PR01256B Otx 1 transcriptionSHHHHHHHHH factor signature II HHH (222-234) 639 2.62−e09 5.92 PR01256BOtx 1 transcription GGPHTSHHHH factor signature II HHLH (217-229) 6403.14e−09 11.65 IPB001541B SUR2-type HHHHHHHHHH hydroxylase/desaturase(223-232) catalytic domain 641 3.14e−09 11.65 IPB001541B SUR2-typeHHHHHHHHHH hydroxylase/desaturase (224-233) catalytic domain 6423.14e−09 11.65 IPB001541B SUR2-type HHHHHHHHHH hydroxylase/desaturase(225-234) catalytic domain 643 6.57e−09 11.65 IPB001541B SUR2-typeHHHHHHHHHH hydroxylase/desaturase (222-231) catalytic domain 6442.29e−08 11.65 IPB00154IB SUR2-type HHHRHHHQSR hydroxylase/desaturase(228-237) catalytic domain 645 3.57e−08 11.65 IPB001541B SUR2-typeHHHHHHHHQS hydroxylase/ (227-236) desaturase catalytic domain 6466.60e−08 0.00 PR00049D Wilm's tumour GSPGPAPAPTP protein signature CPGA(754-768) IV 647 6.61e−08 9.10 PR00334B HMW kininogen GGPHTSHHHHsignature H HHHHHHHHQS RHGK (217-240) 648 6.85e−08 5.92 PR01256B Otx 1transcription HHHHHHHHHH factor signature II HHQ (223-235) 649 7.34e−0814.85 IPB002489C Domain of unknown RFCAPRAGLGH function DUF14ISPEGPLSLSEG PSVGPEGGPAG (46-79) 650 7.75e−08 11.65 IPB001541B SUR2-typeGPHTSHHHHH hydroxylase/desaturase (218-227) catalytic domain 6517.77e−08 3.45 PR01131B Connexin36 (Cx36) GPKAEGRGGS signature II GGD(197-209) 652 8.01e−08 24.91 IPB000868B Isochorismatase HTSHHHHHHHhydrolase family HHHHHQSRHG KRS (220-242) 653 8.32e−08 10.49 PR01274AMetalloprotease TAFPVPTFQDL inhibitor AGFWDL signature I (862-878)

The polypeptides of the invention may play a role in the formation andfunction of the nervous system, by regulating the molecular organizationof synapses and neuronal cell signaling. For example, they couldfunction as adapter proteins linking ion channels and other synapticproteins to the subsynaptic cytoskeleton which is important for thelocalization and concentration of synaptic molecules to the postsynapticmembrane.

The polypeptides, polynucleotides, antibodies and other compositions ofthe invention are expected to be useful in treating the followingdisorders: Alzheimer's disease, anxiety, autism, brain injury,depression, epilepsy, Huntington's disease, mania, pain, Parkinsonism,Parkinson's disease, Schizophrenia, Tardive dyskinesia, myastheniagravis, amyotrophic lateral sclerosis, episodic ataxia/myokymia,hyperkalemix periodic paralysis, hypokalemic periodic paralysis,Lamber-Eaton syndrome, paramyotonia congenita, Rasmussen's encephalitis,Startle disease, and seizure disorders, including neonatal seizuredisorders and generally, learning and memory disorders.

4.11 Definitions

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an” and “the” include plural references unless thecontext clearly dictates otherwise.

The term “active” refers to those forms of the polypeptide that retainthe biologic and/or immunologic activities of any naturally occurringpolypeptide. According to the invention, the terms “biologically active”or “biological activity” refer to a protein or peptide havingstructural, regulatory or biochemical functions of a naturally occurringmolecule. Likewise “biologically active” or “biological activity” refersto the capability of the natural, recombinant or synthetic polypeptideof the invention, or any peptide thereof, to induce a specificbiological response in appropriate animals or cells and to bind withspecific antibodies.

The term “activated cells” as used in this application are those cellswhich are engaged in extracellular or intracellular membranetrafficking, including the export of secretory or enzymatic molecules aspart of a normal or disease process.

The terms “complementary” or “complementarity” refer to the naturalbinding of polynucleotides by base pairing. For example, the sequence5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′. Complementaritybetween two single-stranded molecules may be “partial” such that onlysome of the nucleic acids bind or it may be “complete” such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between the nucleic acid strands has significanteffects on the efficiency and strength of the hybridization between thenucleic acid strands.

The term “embryonic stem cells (ES)” refers to a cell that can give riseto many differentiated cell types in an embryo or an adult, includingthe germ cells. The term “germ line stem cells (GSCs)” refers to stemcells derived from primordial stem cells that provide a steady andcontinuous source of germ cells for the production of gametes. The term“primordial germ cells (PGCs)” refers to a small population of cells setaside from other cell lineages particularly from the yolk sac,mesenteries, or gonadal ridges during embryogenesis that have thepotential to differentiate into germ cells and other cells. PGCs are thesource from which GSCs and ES cells are derived. The PGCs, the GSCs andthe ES cells are capable of self-renewal. Thus these cells not onlypopulate the germ line and give rise to a plurality of terminallydifferentiated cells that comprise the adult specialized organs, but areable to regenerate themselves. The term “totipotent” refers to thecapability of a cell to differentiate into all of the cell types of anadult organism. The term “pluripotent” refers to the capability of acell to differentiate into a number of differentiated cell types thatare present in an adult organism. A pluripotent cell is restricted inits differentiation capability in comparison to a totipotent cell.

The term “expression modulating fragment,” EMF, means a series ofnucleotides that modulates the expression of an operably linked ORF oranother EMF.

As used herein, a sequence is said to “modulate the expression of anoperably linked sequence” when the expression of the sequence is alteredby the presence of the EMF. EMFs include, but are not limited to,promoters, and promoter modulating sequences (inducible elements). Oneclass of EMFs is nucleic acid fragments which induce the expression ofan operably linked ORF in response to a specific regulatory factor orphysiological event.

The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or“oligonculeotide” are used interchangeably and refer to a heteropolymerof nucleotides or the sequence of these nucleotides. These phrases alsorefer to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA) or to any DNA-like orRNA-like material. In the sequences, A is adenine, C is cytosine, G isguanine, and T is thymine, while N is A, T, G, or C. It is contemplatedthat where the polynucleotide is RNA, the T (thymine) in the sequenceherein may be replaced with U (uracil). Generally, nucleic acid segmentsprovided by this invention may be assembled from fragments of the genomeand short oligonucleotide linkers, or from a series of oligonucleotides,or from individual nucleotides, to provide a synthetic nucleic acidwhich is capable of being expressed in a recombinant transcriptionalunit comprising regulatory elements derived from a microbial or viraloperon, or a eukaryotic gene.

The terms “oligonucleotide fragment” or a “polynucleotide fragment”,“portion,” or “segment” or “probe” or “primer” are used interchangeablyand refer to a sequence of nucleotide residues which are at least about5 nucleotides, more preferably at least about 7 nucleotides, morepreferably at least about 9 nucleotides, more preferably at least about11 nucleotides and most preferably at least about 17 nucleotides. Thefragment is preferably less than about 500 nucleotides, preferably lessthan about 200 nucleotides, more preferably less than about 100nucleotides, more preferably less than about 50 nucleotides and mostpreferably less than 30 nucleotides. Preferably the probe is from about6 nucleotides to about 200 nucleotides, preferably from about 15 toabout 50 nucleotides, more preferably from about 17 to 30 nucleotidesand most preferably from about 20 to 25 nucleotides. Preferably thefragments can be used in polymerase chain reaction (PCR), varioushybridization procedures or microarray procedures to identify or amplifyidentical or related parts of mRNA or DNA molecules. A fragment orsegment may uniquely identify each polynucleotide sequence of thepresent invention. Preferably the fragment comprises a sequencesubstantially similar to a portion of SEQ ID NO: 1-4, 6, 14, 16, 25-27,29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301,303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409,418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517,526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578, 580, 587, 589,601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631.

Probes may, for example, be used to determine whether specific mRNAmolecules are present in a cell or tissue or to isolate similar nucleicacid sequences from chromosomal DNA as described by Walsh et al. (Walsh,P. S. et al., PCR Methods Appl. 1:241-250 (1992)). They may be labeledby nick translation, Klenow fill-in reaction, PCR, or other methods wellknown in the art. Probes of the present invention, their preparationand/or labeling are elaborated in Sambrook, J. et al., 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; orAusubel, F. M. et al., 1989, Current Protocols in Molecular Biology,John Wiley & Sons, New York N.Y., both of which are incorporated hereinby reference in their entirety.

The nucleic acid sequences of the present invention also include thesequence information from any of the nucleic acid sequences of SEQ IDNO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216,240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356,377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504,506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573,577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621,623, 625, 627, 629, or 631. The sequence information can be a segment ofSEQ ID NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214,216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354,356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503,504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573,577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621,623, 625, 627, 629, or 631 that uniquely identifies or represents thesequence information of SEQ ID NO:1-4,6,14,16,25-27,29,157-159,161,183-185, 187, 214, 216, 240, 242, 271,273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379,405-407, 409, 418-419,421,441-443, 485-486, 488, 503, 504, 506, 514-515,517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578, 580, 587,589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or631. One such segment can be a twenty-mer nucleic acid sequence becausethe probability that a twenty-mer is fully matched in the human genomeis 1 in 300. In the human genome, there are three billion base pairs inone set of chromosomes. Because 4²⁰ possible twenty-mers exist, thereare 300 times more twenty-mers than there are base pairs in a set ofhuman chromosomes. Using the same analysis, the probability for aseventeen-mer to be fully matched in the human genome is approximately 1in 5. When these segments are used in arrays for expression studies,fifteen-mer segments can be used. The probability that the fifteen-meris fully matched in the expressed sequences is also approximately one infive because expressed sequences comprise less than approximately 5% ofthe entire genome sequence.

Similarly, when using sequence information for detecting a singlemismatch, a segment can be a twenty-five mer. The probability that thetwenty-five mer would appear in a human genome with a single mismatch iscalculated by multiplying the probability for a full match (1÷4²⁵) timesthe increased probability for mismatch at each nucleotide position(3×25). The probability that an eighteen mer with a single mismatch canbe detected in an array for expression studies is approximately one infive. The probability that a twenty-mer with a single mismatch can bedetected in a human genome is approximately one in five.

The term “open reading frame,” ORF, means a series of nucleotidetriplets coding for amino acids without any termination codons and is asequence translatable into protein.

The terms “operably linked” or “operably associated” refer tofunctionally related nucleic acid sequences. For example, a promoter isoperably associated or operably linked with a coding sequence if thepromoter controls the transcription of the coding sequence. Whileoperably linked nucleic acid sequences can be contiguous and in the samereading frame, certain genetic elements e.g. repressor genes are notcontiguously linked to the coding sequence but still controltranscription/translation of the coding sequence.

The term “pluripotent” refers to the capability of a cell todifferentiate into a number of differentiated cell types that arepresent in an adult organism. A pluripotent cell is restricted in itsdifferentiation capability in comparison to a totipotent cell.

The terms “polypeptide” or “peptide” or “amino acid sequence” refer toan oligopeptide, peptide, polypeptide, or protein sequence or fragmentthereof and to naturally occurring or synthetic molecules. A polypeptide“fragment,” “portion,” or “segment” is a stretch of amino acid residuesof at least about 5 amino acids, preferably at least about 7 aminoacids, more preferably at least about 9 amino acids and most preferablyat least about 17 or more amino acids. The peptide preferably is notgreater than about 200 amino acids, more preferably less than 150 aminoacids and most preferably less than 100 amino acids. Preferably thepeptide is from about 5 to about 200 amino acids. To be active, anypolypeptide must have sufficient length to display biological and/orimmunological activity.

The term “naturally occurring polypeptide” refers to polypeptidesproduced by cells that have not been genetically engineered andspecifically contemplates various polypeptides arising frompost-translational modifications of the polypeptide including, but notlimited to, acetylation, carboxylation, glycosylation, phosphorylation,lipidation and acylation.

The term “translated protein coding portion” means a sequence whichencodes for the full length protein which may include any leadersequence or a processing sequence.

The term “mature protein coding sequence” refers to a sequence whichencodes a peptide or protein without any leader/signal sequence. The“mature protein portion” refers to that portion of the protein withoutthe leader/signal sequence. The peptide may have the leader sequencesremoved during processing in the cell or the protein may have beenproduced synthetically or using a polynucleotide only encoding for themature protein coding sequence. It is contemplated that the matureprotein portion may or may not include an initial methionine residue.The initial methionine is often removed during processing of thepeptide.

The term “derivative” refers to polypeptides chemically modified by suchtechniques as ubiquitination, labeling (e.g., with radionuclides orvarious enzymes), covalent polymer attachment such as pegylation(derivatization with polyethylene glycol) and insertion or substitutionby chemical synthesis of amino acids such as ornithine, which do notnormally occur in human proteins.

The term “variant” (or “analog”) refers to any polypeptide differingfrom naturally occurring polypeptides by amino acid insertions,deletions, and substitutions, created using, e.g., recombinant DNAtechniques. Guidance in determining which amino acid residues may bereplaced, added or deleted without abolishing activities of interest,may be found by comparing the sequence of the particular polypeptidewith that of homologous peptides and minimizing the number of amino acidsequence changes made in regions of high homology (conserved regions) orby replacing amino acids with consensus sequence.

Alternatively, recombinant variants encoding these same or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsin the polynucleotide sequence may be reflected in the polypeptide ordomains of other peptides added to the polypeptide to modify theproperties of any part of the polypeptide, to change characteristicssuch as ligand-binding affinities, interchain affinities, ordegradation/turnover rate.

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements.“Conservative” amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Insertions” or “deletions” are preferably in the rangeof about 1 to 20 amino acids, more preferably 1 to 10 amino acids. Thevariation allowed may be experimentally determined by systematicallymaking insertions, deletions, or substitutions of amino acids in apolypeptide molecule using recombinant DNA techniques and assaying theresulting recombinant variants for activity.

Alternatively, where alteration of function is desired, insertions,deletions or non-conservative alterations can be engineered to producealtered polypeptides. Such alterations can, for example, alter one ormore of the biological functions or biochemical characteristics of thepolypeptides of the invention. For example, such alterations may changepolypeptide characteristics such as ligand-binding affinities,interchain affinities, or degradation/turnover rate. Further, suchalterations can be selected so as to generate polypeptides that arebetter suited for expression, scale up and the like in the host cellschosen for expression. For example, cysteine residues can be deleted orsubstituted with another amino acid residue in order to eliminatedisulfide bridges.

The terms “purified” or “substantially purified” as used herein denotesthat the indicated nucleic acid or polypeptide is present in thesubstantial absence of other biological macromolecules, e.g.,polynucleotides, proteins, and the like. In one embodiment, thepolynucleotide or polypeptide is purified such that it constitutes atleast 95% by weight, more preferably at least 99% by weight, of theindicated biological macromolecules present (but water, buffers, andother small molecules, especially molecules having a molecular weight ofless than 1000 daltons, can be present).

The term “isolated” as used herein refers to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) present with the nucleic acid or polypeptide in itsnatural source. In one embodiment, the nucleic acid or polypeptide isfound in the presence of (if anything) only a solvent, buffer, ion, orother components normally present in a solution of the same. The terms“isolated” and “purified” do not encompass nucleic acids or polypeptidespresent in their natural source.

The term “recombinant,” when used herein to refer to a polypeptide orprotein, means that a polypeptide or protein is derived from recombinant(e.g., microbial, insect, or mammalian) expression systems. “Microbial”refers to recombinant polypeptides or proteins made in bacterial orfungal (e.g., yeast) expression systems. As a product, “recombinantmicrobial” defines a polypeptide or protein essentially free of nativeendogenous substances and unaccompanied by associated nativeglycosylation. Polypeptides or proteins expressed in most bacterialcultures, e.g., E. coli, will be free of glycosylation modifications;polypeptides or proteins expressed in yeast will have a glycosylationpattern in general different from those expressed in mammalian cells.

The term “recombinant expression vehicle or vector” refers to a plasmidor phage or virus or vector, for expressing a polypeptide from a DNA(RNA) sequence. An expression vehicle can comprise a transcriptionalunit comprising an assembly of (1) a genetic element or elements havinga regulatory role in gene expression, for example, promoters orenhancers, (2) a structural or coding sequence which is transcribed intomRNA and translated into protein, and (3) appropriate transcriptioninitiation and termination sequences. Structural units intended for usein yeast or eukaryotic expression systems preferably include a leadersequence enabling extracellular secretion of translated protein by ahost cell. Alternatively, where recombinant protein is expressed withouta leader or transport sequence, it may include an amino terminalmethionine residue. This residue may or may not be subsequently cleavedfrom the expressed recombinant protein to provide a final product.

The term “recombinant expression system” means host cells which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit extrachromosomally.Recombinant expression systems as defined herein will expressheterologous polypeptides or proteins upon induction of the regulatoryelements linked to the DNA segment or synthetic gene to be expressed.This term also means host cells which have stably integrated arecombinant genetic element or elements having a regulatory role in geneexpression, for example, promoters or enhancers. Recombinant expressionsystems as defined herein will express polypeptides or proteinsendogenous to the cell upon induction of the regulatory elements linkedto the endogenous DNA segment or gene to be expressed. The cells can beprokaryotic or eukaryotic.

The term “secreted” includes a protein that is transported across orthrough a membrane, including transport as a result of signal sequencesin its amino acid sequence when it is expressed in a suitable host cell.“Secreted” proteins include without limitation proteins secreted wholly(e.g., soluble proteins) or partially (e.g., receptors) from the cell inwhich they are expressed. “Secreted” proteins also include withoutlimitation proteins that are transported across the membrane of theendoplasmic reticulum. “Secreted” proteins are also intended to includeproteins containing non-typical signal sequences (e.g. Interleukin-1Beta, see Krasney, P. A. and Young, P. R. Cytokine 4:134-143 (1992)) andfactors released from damaged cells (e.g. Interleukin-1 ReceptorAntagonist, see Arend, W. P. et. al. Annu. Rev. Immunol. 16:27-55(1998)).

Where desired, an expression vector may be designed to contain a “signalor leader sequence” which will direct the polypeptide through themembrane of a cell. Such a sequence may be naturally present on thepolypeptides of the present invention or provided from heterologousprotein sources by recombinant DNA techniques.

The term “stringent” is used to refer to conditions that are commonlyunderstood in the art as stringent. Stringent conditions can includehighly stringent conditions (i.e., hybridization to filter-bound DNA in0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringentconditions (ie., washing in 0.2×SSC/0.1% SDS at 42° C.). Other exemplaryhybridization conditions are described herein in the examples.

In instances of hybridization of deoxyoligonucleotides, additionalexemplary stringent hybridization conditions include washing in6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-baseoligonucleotides), 48° C. (for 17-base oligonucleotides), 55° C. (for20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).

As used herein, “substantially equivalent” can refer both to nucleotideand amino acid sequences, for example a mutant sequence, that variesfrom a reference sequence by one or more substitutions, deletions, oradditions, the net effect of which does not result in an adversefunctional dissimilarity between the reference and subject sequences.Typically, such a substantially equivalent sequence varies from one ofthose listed herein by no more than about 35% (i.e., the number ofindividual residue substitutions, additions, and/or deletions in asubstantially equivalent sequence, as compared to the correspondingreference sequence, divided by the total number of residues in thesubstantially equivalent sequence is about 0.35 or less). Such asequence is said to have 65% sequence identity to the listed sequence.In one embodiment, a substantially equivalent, e.g., mutant, sequence ofthe invention varies from a listed sequence by no more than 30% (70%sequence identity); in a variation of this embodiment, by no more than25% (75% sequence identity); and in a further variation of thisembodiment, by no more than 20% (80% sequence identity) and in a furthervariation of this embodiment, by no more than 10% (90% sequenceidentity) and in a further variation of this embodiment, by no more that5% (95% sequence identity). Substantially equivalent, e.g., mutant,amino acid sequences according to the invention preferably have at least80% sequence identity with a listed amino acid sequence, more preferablyat least 90% sequence identity. Substantially equivalent nucleotidesequence of the invention can have lower percent sequence identities,taking into account, for example, the redundancy or degeneracy of thegenetic code. Preferably, nucleotide sequence has at least about 65%identity, more preferably at least about 75% identity, and mostpreferably at least about 95% identity. For the purposes of the presentinvention, sequences having substantially equivalent biological activityand substantially equivalent expression characteristics are consideredsubstantially equivalent. For the purposes of determining equivalence,truncation of the mature sequence (e.g., via a mutation which creates aspurious stop codon) should be disregarded. Sequence identity may bedetermined, e.g., using the Jotun Hein method (Hein, J. Methods Enzymol.183:626-645 (1990)). Identity between sequences can also be determinedby other methods known in the art, e.g. by varying hybridizationconditions.

The term “totipotent” refers to the capability of a cell todifferentiate into all of the cell types of an adult organism.

The term “transformafion” means introducing DNA into a suitable hostcell so that the DNA is replicable, either as an extrachromosomalelement, or by chromosomal integration. The term “transfection” refersto the taking up of an expression vector by a suitable host cell,whether or not any coding sequences are in fact expressed. The term“infection” refers to the introduction of nucleic acids into a suitablehost cell by use of a virus or viral vector.

As used herein, an “uptake modulating fragment,” UMF, means a series ofnucleotides which mediate the uptake of a linked DNA fragment into acell. UMFs can be readily identified using known UMFs as a targetsequence or target motif with the computer-based systems describedbelow. The presence and activity of a UMF can be confirmed by attachingthe suspected UMF to a marker sequence. The resulting nucleic acidmolecule is then incubated with an appropriate host under appropriateconditions and the uptake of the marker sequence is determined. Asdescribed above, a UMF will increase the frequency of uptake of a linkedmarker sequence.

Each of the above terms is meant to encompass all that is described foreach, unless the context dictates otherwise.

4.12 Nucleic Acids of the Invention

The isolated polynucleotides of the invention include, but are notlimited to a polynucleotide comprising any of the nucleotide sequencesof SEQ ID NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187,214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349,353-354, 356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486,488, 503, 504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558,570-571, 573, 577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617,619, 621, 623, 625, 627, 629, or 631; a fragment of SEQ ID NO: 1-4, 6,14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271,273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379,405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506,514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578,580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625,627, 629, or 631; a polynucleotide comprising the full length proteincoding sequence of SEQ ID NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161,183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324,345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421,441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547,549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606, 608,611, 613, 617, 619, 621, 623, 625, 627, 629, or 631 (for example codingfor SEQ ID NO: 5, 15, 28, 160, 186, 215, 241, 272, 302, 323, 348, 355,378, 408, 420, 444, 487, 505, 516, 528, 542, 548, 557, 572, 579, 588,602, 607, 612, 618, 622, 626, or 630); and a polynucleotide comprisingthe nucleotide sequence encoding the mature protein coding sequence ofthe polypeptides of any one of SEQ ID NO: 5, 7-13, 15, 17-24, 28,30-156, 160, 162-182, 186, 188-213, 215, 217-239, 241, 243-270, 272,274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355, 357-376, 378,380-401, 408, 410-414, 415, 420, 422-439, 444-480,482-484,487, 489-501,505, 507-512, 516, 518-524, 528, 530-539, 542, 544-546, 548, 550-553,557, 559-567, 572, 574, 576, 579, 581-584, 588, 590, 596, 602, 604-605,607, 609-610, 612, 614-615, 618, 620, 622, 624, 626, 628, 630, 632, or634-653. The polynucleotides of the present invention also include, butare not limited to, a polynucleotide that hybridizes under stringentconditions to (a) the complement of any of the nucleotides sequences ofSEQ ID NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214,216, 240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354,356, 377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503,504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573,577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621,623, 625, 627, 629, or 631; (b) a polynucleotide encoding any one of thepolypeptides of SEQ ID NO: 5, 7-13, 15, 17-24, 28, 30-156, 160, 162-182,186, 188-213, 215, 217-239, 241, 243-270, 272, 274-299, 302, 304-321,323, 325-344, 348, 350-352, 355, 357-376, 378, 380-401, 408, 410-414,415, 420, 422-439, 444-480, 482-484, 487, 489-501, 505, 507-512, 516,518-524, 528, 530-539, 542, 544-546, 548, 550-553, 557, 559-567, 572,574, 576, 579, 581-584, 588, 590, 596, 602, 604-605, 607, 609-610, 612,614-615, 618, 620, 622, 624, 626, 628, 630, 632, or 634-653; (c) apolynucleotide which is an allelic variant of any polynucleotidesrecited above; (d) a polynucleotide which encodes a species homolog ofany of the proteins recited above; or (e) a polynucleotide that encodesa polypeptide comprising a specific domain or truncation of thepolypeptides of SEQ ID NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161,183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324,345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421,441-443, 485486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547,549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606, 608,611, 613, 617, 619, 621, 623, 625, 627, 629, or 631. Domains of interestmay depend on the nature of the encoded polypeptide; e.g., domains inreceptor-like polypeptides include ligand-binding, extracellular,transmembrane, or cytoplasmic domains, or combinations thereof; domainsin immunoglobulin-like proteins include the variable immunoglobulin-likedomains; domains in enzyme-like polypeptides include catalytic andsubstrate binding domains; and domains in ligand polypeptides includereceptor-binding domains.

The polynucleotides of the invention include naturally occurring orwholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA,e.g., mRNA. The polynucleotides may include the entire coding region ofthe cDNA or may represent a portion of the coding region of the cDNA.

The present invention also provides genes corresponding to the cDNAsequences disclosed herein. The corresponding genes can be isolated inaccordance with known methods using the sequence information disclosedherein. Such methods include the preparation of probes or primers fromthe disclosed sequence information for identification and/oramplification of genes in appropriate genomic libraries or other sourcesof genomic materials. Further 5′ and 3′ sequence can be obtained usingmethods known in the art. For example, full length cDNA or genomic DNAthat corresponds to any of the polynucleotides of SEQ ID NO: 1-4, 6, 14,16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273,300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407,409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517,526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578, 580, 587, 589,601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631can be obtained by screening appropriate cDNA or genomic DNA librariesunder suitable hybridization conditions using any of the polynucleotidesof SEQ ID NO: 1-4,6,14,16,25-27,29,157-159,161,183-185, 187, 214, 216,240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356,377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504,506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573,577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621,623, 625, 627, 629, or 631 or a portion thereof as a probe.Alternatively, the polynucleotides of SEQ ID NO:1-4,6,14,16,25-27,29,157-159,161,183-185, 187, 214, 216, 240, 242, 271,273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379,405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506,514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578,580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625,627, 629, or 631 may be used as the basis for suitable primer(s) thatallow identification and/or amplification of genes in appropriategenomic DNA or cDNA libraries.

The nucleic acid sequences of the invention can be assembled from ESTsand sequences (including cDNA and genomic sequences) obtained from oneor more public databases, such as dbEST, gbpri, and UniGene. The ESTsequences can provide identifying sequence information, representativefragment or segment information, or novel segment information for thefull-length gene.

The polynucleotides of the invention also provide polynucleotidesincluding nucleotide sequences that are substantially equivalent to thepolynucleotides recited above. Polynucleotides according to theinvention can have, e.g., at least about 65%, at least about 70%, atleast about 75%, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, or 89%, more typically at least about 90%, 91%, 92%, 93%, or 94%and even more typically at least about 95%, 96%, 97%, 98% or 99%sequence identity to a polynucleotide recited above.

Included within the scope of the nucleic acid sequences of the inventionare nucleic acid sequence fragments that hybridize under stringentconditions to any of the nucleotide sequences of SEQ ID NO: 1-4, 6, 14,16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273,300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407,409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517,526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578,580,587, 589,601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631,or complements thereof, which fragment is greater than about 5nucleotides, preferably 7 nucleotides, more preferably greater than 9nucleotides and most preferably greater than 17 nucleotides. Fragmentsof, e.g. 15, 17, or 20 nucleotides or more that are selective for (i.e.specifically hybridize to any one of the polynucleotides of theinvention) are contemplated. Probes capable of specifically hybridizingto a polynucleotide can differentiate polynucleotide sequences of theinvention from other polynucleotide sequences in the same family ofgenes or can differentiate human genes from genes of other species, andare preferably based on unique nucleotide sequences.

The sequences falling within the scope of the present invention are notlimited to these specific sequences, but also include allelic andspecies variations thereof. Allelic and species variations can beroutinely determined by comparing the sequence provided in SEQ ID NO:1-4,6,14,16,25-27,29,157-159,161,183-185, 187, 214, 216, 240, 242, 271,273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379,405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506,514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578,580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625,627, 629, or 631, a representative fragment thereof, or a nucleotidesequence at least 90% identical, preferably 95% identical, to SEQ ID NO:1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240,242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377,379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506,514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578,580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625,627, 629, or 631 with a sequence from another isolate of the samespecies. Furthermore, to accommodate codon variability, the inventionincludes nucleic acid molecules coding for the same amino acid sequencesas do the specific ORFs disclosed herein. In other words, in the codingregion of an ORF, substitution of one codon for another codon thatencodes the same amino acid is expressly contemplated.

The nearest neighbor result for the nucleic acids of the presentinvention, including SEQ ID NO: 14, 6, 14, 16, 25-27, 29, 157-159, 161,183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324,345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421,441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547,549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606, 608,611, 613, 617, 619, 621, 623, 625, 627, 629, or 631, can be obtained bysearching a database using an algorithm or a program. Preferably, aBLAST which stands for Basic Local Alignment Search Tool is used tosearch for local sequence alignments (Altshul, S. F., J. Mol. Evol. 36290-300 (1993) and Altschul S. F., et al. J. Mol. Biol. 21:403-410(1990)).

Species homologs (or orthologs) of the disclosed polynucleotides andproteins are also provided by the present invention. Species homologsmay be isolated and identified by making suitable probes or primers fromthe sequences provided herein and screening a suitable nucleic acidsource from the desired species.

The invention also encompasses allelic variants of the disclosedpolynucleotides or proteins; that is, naturally-occurring alternativeforms of the isolated polynucleotide which also encodes proteins whichare identical, homologous or related to that encoded by thepolynucleotides.

The nucleic acid sequences of the invention are further directed tosequences which encode variants of the described nucleic acids. Theseamino acid sequence variants may be prepared by methods known in the artby introducing appropriate nucleotide changes into a native or variantpolynucleotide. There are two variables in the construction of aminoacid sequence variants: the location of the mutation and the nature ofthe mutation. Nucleic acids encoding the amino acid sequence variantsare preferably constructed by mutating the polynucleotide to encode anamino acid sequence that does not occur in nature. These nucleic acidalterations can be made at sites that differ in the nucleic acids fromdifferent species (variable positions) or in highly conserved regions(constant regions). Sites at such locations will typically be modifiedin series, e.g., by substituting first with conservative choices (e.g.,hydrophobic amino acid to a different hydrophobic amino acid) and thenwith more distant choices (e.g., hydrophobic amino acid to a chargedamino acid), and then deletions or insertions may be made at the targetsite. Amino acid sequence deletions generally range from about 1 to 30residues, preferably about 1 to 10 residues, and are typicallycontiguous. Amino acid insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one to one hundred ormore residues, as well as intrasequence insertions of single or multipleamino acid residues. Intrasequence insertions may range generally fromabout 1 to 10 amino residues, preferably from 1 to 5 residues. Examplesof terminal insertions include the heterologous signal sequencesnecessary for secretion or for intracellular targeting in different hostcells and sequences such as FLAG or poly-histidine sequences useful forpurifying the expressed protein.

In a preferred method, polynucleotides encoding the novel amino acidsequences are changed via site-directed mutagenesis. This method usesoligonucleotide sequences to alter a polynucleotide to encode thedesired amino acid variant, as well as sufficient adjacent nucleotideson both sides of the changed amino acid to form a stable duplex oneither side of the site being changed. In general, the techniques ofsite-directed mutagenesis are well known to those of skill in the artand this technique is exemplified by publications such as, Edelman etal., DNA 2:183 (1983). A versatile and efficient method for producingsite-specific changes in a polynucleotide sequence was published byZoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may alsobe used to create amino acid sequence variants of the novel nucleicacids. When small amounts of template DNA are used as starting material,primer(s) that differs slightly in sequence from the correspondingregion in the template DNA can generate the desired amino acid variant.PCR amplification results in a population of product DNA fragments thatdiffer from the polynucleotide template encoding the polypeptide at theposition specified by the primer. The product DNA fragments replace thecorresponding region in the plasmid and this gives a polynucleotideencoding the desired amino acid variant.

A further technique for generating amino acid variants is the cassettemutagenesis technique described in Wells, et al., Gene 34:315 (1985);and other mutagenesis techniques well known in the art, such as, forexample, the techniques in Sambrook, et al., supra, and CurrentProtocols in Molecular Biology, Ausubel, et al. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be used in the practice of the invention for the cloning andexpression of these novel nucleic acids. Such DNA sequences includethose which are capable of hybridizing to the appropriate novel nucleicacid sequence under stringent conditions.

Polynucleotides encoding preferred polypeptide truncations of theinvention can be used to generate polynucleotides encoding chimeric orfusion proteins comprising one or more domains of the invention andheterologous protein sequences.

The polynucleotides of the invention additionally include the complementof any of the polynucleotides recited above. The polynucleotide can beDNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods andalgorithms for obtaining such polynucleotides are well known to those ofskill in the art and can include, for example, methods for determininghybridization conditions that can routinely isolate polynucleotides ofthe desired sequence identities.

In accordance with the invention, polynucleotide sequences comprisingthe mature protein coding sequences, coding for any one of SEQ ID NO: 5,15, 28, 160, 186, 215, 241, 272, 302, 323, 348, 355, 378, 408, 420, 444,487, 505, 516, 528, 542, 548, 557, 572, 579, 588, 602, 607, 612, 618,622, 626, or 630, or functional equivalents thereof, may be used togenerate recombinant DNA molecules that direct the expression of thatnucleic acid, or a functional equivalent thereof, in appropriate hostcells. Also included are the cDNA inserts of any of the clonesidentified herein.

A polynucleotide according to the invention can be joined to any of avariety of other nucleotide sequences by well-established recombinantDNA techniques (see Sambrook, J. et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotidesequences for joining to polynucleotides include an assortment ofvectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids,and the like, that are well known in the art. Accordingly, the inventionalso provides a vector including a polynucleotide of the invention and ahost cell containing the polynucleotide. In general, the vector containsan origin of replication functional in at least one organism, convenientrestriction endonuclease sites, and a selectable marker for the hostcell. Vectors according to the invention include expression vectors,replication vectors, probe generation vectors, and sequencing vectors. Ahost cell according to the invention can be a prokaryotic or eukaryoticcell and can be a unicellular organism or part of a multicellularorganism.

The present invention further provides recombinant constructs comprisinga nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-4,6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242,271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379,405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506,514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578,580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625,627, 629, or 631 or a fragment thereof or any other polynucleotides ofthe invention. In one embodiment, the recombinant constructs of thepresent invention comprise a vector, such as a plasmid or viral vector,into which a nucleic acid having any of the nucleotide sequences of SEQID NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216,240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356,377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504,506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573,577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621,623, 625, 627, 629, or 631 or a fragment thereof is inserted, in aforward or reverse orientation. In the case of a vector comprising oneof the ORFs of the present invention, the vector may further compriseregulatory sequences, including for example, a promoter, operably linkedto the ORF. Large numbers of suitable vectors and promoters are known tothose of skill in the art and are commercially available for generatingthe recombinant constructs of the present invention. The followingvectors are provided by way of example. Bacterial: pBs, phagescript,PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a(Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV,pMSG, and pSVL (Pharmacia).

The isolated polynucleotide of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids Res. 19:4485-4490(1991), in order to produce the protein recombinantly. Many suitableexpression control sequences are known in the art. General methods ofexpressing recombinant proteins are also known and are exemplified in R.Kaufman, Methods in Enzymology 185:537-566 (1990). As defined herein“operably linked” means that the isolated polynucleotide of theinvention and an expression control sequence are situated within avector or cell in such a way that the protein is expressed by a hostcell which has been transformed (transfected) with the ligatedpolynucleotide/expression control sequence.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art. Generally, recombinant expressionvectors will include origins of replication and selectable markerspermitting transformation of the host cell, e.g., the ampicillinresistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoterderived from a highly expressed gene to direct transcription of adownstream structural sequence. Such promoters can be derived fromoperons encoding glycolytic enzymes such as 3-phosphoglycerate kinase(PGK), a-factor, acid phosphatase, or heat shock proteins, among others.The heterologous structural sequence is assembled in appropriate phasewith translation initiation and termination sequences, and preferably, aleader sequence capable of directing secretion of translated proteininto the periplasmic space or extracellular medium. Optionally, theheterologous sequence can encode a fusion protein including an aminoterminal identification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct. Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed. Followingtransformation of a suitable host strain and growth of the host strainto an appropriate cell density, the selected promoter is induced orderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification.

Polynucleotides of the invention can also be used to induce immuneresponses. For example, as described in Fan, et al., Nat. Biotech.17:870-872 (1999), incorporated herein by reference, nucleic acidsequences encoding a polypeptide may be used to generate antibodiesagainst the encoded polypeptide following topical administration ofnaked plasmid DNA or following injection, and preferably intramuscularinjection of the DNA. The nucleic acid sequences are preferably insertedin a recombinant expression vector and may be in the form of naked DNA.

4.12.1 Antisense Nucleic Acids

Another aspect of the invention pertains to isolated antisense nucleicacid molecules that can hybridize to or are complementary to the nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO: 1-4, 6,14, 16, 25-27,29,157-159, 161, 183-185, 187, 214, 216, 240, 242, 271,273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379,405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506,514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578,580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625,627, 629, or 631, or fragments, analogs or derivatives thereof. An“antisense” nucleic acid comprises a nucleotide sequence that iscomplementary to a “sense” nucleic acid encoding a protein (e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence). In specific aspects, antisensenucleic acid molecules are provided that comprise a sequencecomplementary to at least about 10, 25, 50, 100, 250 or 500 nucleotidesor an entire coding strand, or to only a portion thereof. Nucleic acidmolecules encoding fragments, homologs, derivatives and analogs of aprotein of any of SEQ ID NO: 5, 7-13, 15, 17-24, 28, 30-156, 160,162-182, 186, 188-213, 215, 217-239, 241, 243-270, 272, 274-299, 302,304-321, 323, 325-344, 348, 350-352, 355, 357-376, 378, 380-401, 408,410414, 415, 420, 422-439, 444-480, 482-484, 487, 489-501, 505, 507-512,516, 518-524, 528, 530-539, 542, 544-546, 548, 550-553, 557, 559-567,572, 574, 576, 579, 581-584, 588, 590, 596, 602, 604-605, 607, 609-610,612, 614-615, 618, 620, 622, 624, 626, 628, 630, 632, or 634-653 orantisense nucleic acids complementary to a nucleic acid sequence of SEQID NO: 1-4, 6, 14, 16, 25-27, 29, 157-159,161,183-185, 187, 214, 216,240, 242, 271, 273, 300-301,303, 322, 324, 345-347, 349, 353-354, 356,377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504,506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573,577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621,623, 625, 627, 629, or 631 are additionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence of theinvention. The term “coding region” refers to the region of thenucleotide sequence comprising codons which are translated into aminoacid residues. In another embodiment, the antisense nucleic acidmolecule is antisense to a “conceding region” of the coding strand of anucleotide sequence of the invention. The term “conceding region” refersto 5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

Given the coding strand sequences (e.g. SEQ ID NO: 1-4, 6, 14, 16,25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273,300-301, 303, 322, 324, 345-347, 349,353-354,356,377,379,405-407,409,418-419, 421, 441-443,485-486, 488, 503,504, 506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573,577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621,623, 625, 627, 629, or 631) disclosed herein, antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crickor Hoogsteen base pairing. The antisense nucleic acid molecule can becomplementary to the entire coding region of an mRNA of the invention,but more preferably is an oligonucleotide that is antisense to only aportion of the coding or noncoding region of an mRNA of the invention.For example, the antisense oligonucleotide can be complementary to theregion surrounding the translation start site of an mRNA of theinvention. An antisense oligonucleotide can be, for example, about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. Anantisense nucleic acid of the invention can be constructed usingchemical synthesis or enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids (e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used).

Examples of modified nucleotides that can be used to generate theantisense nucleic acid include: 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following section).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a proteinaccording to the invention to thereby inhibit expression of the protein(e.g., by inhibiting transcription and/or translation). Thehybridization can be by conventional nucleotide complementarity to forma stable duplex, or, for example, in the case of an antisense nucleicacid molecule that binds to DNA duplexes, through specific interactionsin the major groove of the double helix. An example of a route ofadministration of antisense nucleic acid molecules of the inventionincludes direct injection at a tissue site. Alternatively, antisensenucleic acid molecules can be modified to target selected cells and thenadministered systemically. For example, for systemic administration,antisense molecules can be modified such that they specifically bind toreceptors or antigens expressed on a selected cell surface (e.g., bylinking the antisense nucleic acid molecules to peptides or antibodiesthat bind to cell surface receptors or antigens). The antisense nucleicacid molecules can also be delivered to cells using the vectorsdescribed herein. To achieve sufficient nucleic acid molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an alpha-anomeric nucleic acid molecule. An alpha-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual alpha-units, thestrands run parallel to each other. See, e.g., Gaultier, et al., Nucl.Acids Res. 15:6625-6641 (1987). The antisense nucleic acid molecule canalso comprise a 2′-o-methylribonucleotide (see, e.g., Inoue, et al.Nucl. Acids Res. 15:6131-6148 (1987)) or a chimeric RNA-DNA analogue(see, e.g., Inoue, et al., FEBS Lett. 215:327-330 (1987).

4.12.2 Ribozymes And PNA Moieties

Nucleic acid modifications include, by way of non-limiting example,modified bases, and nucleic acids whose sugar phosphate backbones aremodified or derivatized. These modifications are carried out at least inpart to enhance the chemical stability of the modified nucleic acid,such that they can be used, for example, as antisense binding nucleicacids in therapeutic applications in a subject.

In one embodiment, an antisense nucleic acid of the invention is aribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity that are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes as described in Haselhoff andGerlach, Nature 334: 585-591 (1988)) can be used to catalytically cleavemRNA transcripts of the invention to thereby inhibit translation of mRNAof the invention. A ribozyme having specificity for a nucleic acid ofthe invention can be designed based upon the nucleotide sequence of acDNA disclosed herein (e.g. SEQ ID NO: 1-4, 6, 14, 16, 25-27, 29,157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303,322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419,421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529,547, 549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606,608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631). For example,a derivative of a Tetrahymena L-19 IVS RNA can be constructed in whichthe nucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in an mRNA of the invention. See,e.g., U.S. Pat. No. 4,987,071 to Cech, et al. and U.S. Pat. No.5,116,742 to Cech, et al. Stem cell growth factor-like mRNA can also beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel, et al., Science261:1411-1418 (1993).

Alternatively, gene expression can be inhibited by targeting nucleotidesequences complementary to the regulatory region (e.g., the promoterand/or enhancers of the gene relating to the invention) to form triplehelical structures that prevent transcription of the gene in targetcells. See, e.g., Helene, Anticancer Drug Des. 6:569-84 (1991); Helene,et al., Ann. N.Y. Acad. Sci. 660:27-36 (1992); Maher, Bioassays14:807-15 (1992).

In various embodiments, the nucleic acids of the invention can bemodified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids. See, e.g.,Hyrup, et al., Bioorg. Med. Chem. 4:5-23 (1996). As used herein, theterms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics(e.g., DNA mimics) in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrup,et al., 1996. supra; Perry-O'Keefe, et al., Proc. Natl. Acad. Sci. USA93:14670-14675 (1996).

PNAs of the invention can be used in therapeutic and diagnosticapplications. For example, PNAs can be used as antisense or antigeneagents for sequence-specific modulation of gene expression by, e.g.,inducing transcription or translation arrest or inhibiting replication.PNAs of the invention can also be used, for example, in the analysis ofsingle base pair mutations in a gene (e.g., PNA directed PCR clamping;as artificial restriction enzymes when used in combination with otherenzymes, e.g., S1 nucleases (see, Hyrup, et al., 1996.supra); or asprobes or primers for DNA sequence and hybridization (see, Hyrup, etal., 1996, supra; Perry-O'Keefe, et al., 1996. supra).

In another embodiment, PNAs of the invention can be modified, e.g., toenhance their stability or cellular uptake, by attaching lipophilic orother helper groups to PNA, by the formation of PNA-DNA chimeras, or bythe use of liposomes or other techniques of drug delivery known in theart. For example, PNA-DNA chimeras of the invention can be generatedthat may combine the advantageous properties of PNA and DNA. Suchchimeras allow DNA recognition enzymes (e.g., RNase H and DNApolymerases) to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation(see, Hyrup, et al., 1996. supra). The synthesis of PNA-DNA chimeras canbe performed as described in Hyrup, et al., 1996. Supra, et al., NuclAcids Res 24:3357-3363 (1996). For example, a DNA chain can besynthesized on a solid support using standard phosphoramidite couplingchemistry, and modified nucleoside analogs, e.g.,5′-(4-methoxytrityl)amino-5′-deoxythymidine phosphoramidite, can be usedbetween the PNA and the 5′ end of DNA. See, e.g., Mag, et al., Nucl AcidRes 17:5973-5988 (1989). PNA monomers are then coupled in a stepwisemanner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNAsegment. See, e.g., Finn, et al., 1996. supra. Alternatively, chimericmolecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment.See, e.g., Petersen, et al., Bioorg. Med. Chem. Lett. 5:1119-11124(1975).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger, et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556(1989); Lemaitre, et al., Proc. Natl. Acad. Sci. USA 84:648-652 (1987);PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g.,PCT Publication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krol,et al., BioTechniques 6:958-976 (1988)) or intercalating agents (see,e.g., Zon, Pharm. Res. 5:539-549 (1988)). To this end, theoligonucleotide can be conjugated to another molecule, e.g., a peptide,a hybridization triggered cross-linking agent, a transport agent, ahybridization-triggered cleavage agent, and the like.

4.13 Hosts

The present invention further provides host cells genetically engineeredto contain the polynucleotides of the invention. For example, such hostcells may contain nucleic acids of the invention introduced into thehost cell using known transformation, transfection or infection methods.The present invention still further provides host cells geneticallyengineered to express the polynucleotides of the invention, wherein suchpolynucleotides are in operative association with a regulatory sequenceheterologous to the host cell which drives expression of thepolynucleotides in the cell.

The host cell can be a higher eukaryotic host cell, such as a mammaliancell, a lower eukaryotic host cell, such as a yeast cell, or the hostcell can be a prokaryotic cell, such as a bacterial cell. Introductionof the recombinant construct into the host cell can be effected bycalcium phosphate transfection, DEAE, dextran mediated transfection, orelectroporation (Davis, L. et al., Basic Methods in Molecular Biology(1986)). The host cells containing one of polynucleotides of theinvention, can be used in conventional manners to produce the geneproduct encoded by the isolated fragment (in the case of an ORF) or canbe used to produce a heterologous protein under the control of the EMF.

Any host/vector system can be used to express one or more of the ORFs ofthe present invention. These include, but are not limited to, eukaryotichosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9 cells, as wellas prokaryotic host such as E. coli and B. subtilis. The most preferredcells are those which do not normally express the particular polypeptideor protein or which expresses the polypeptide or protein at low naturallevel. Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al., inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989), the disclosure of which is hereby incorporated byreference.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell tines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter, and also any necessary ribosome binding sites,polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking nontranscribed sequences. DNAsequences derived from the SV40 viral genome, for example, SV40 origin,early promoter, enhancer, splice, and polyadenylation sites may be usedto provide the required nontranscribed genetic elements. Recombinantpolypeptides and proteins produced in bacterial culture are usuallyisolated by initial extraction from cell pellets, followed by one ormore salting-out, aqueous ion exchange or size exclusion chromatographysteps. Protein refolding steps can be used, as necessary, in completingconfiguration of the mature protein. Finally, high performance liquidchromatography (HPLC) can be employed for final purification steps.Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents.

A number of types of cells may act as suitable host cells for expressionof the protein. Mammalian host cells include, for example, monkey COScells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, humanepidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, othertransformed primate cell lines, normal diploid cells, cell strainsderived from in vitro culture of primary tissue, primary explants, HeLacells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.

Alternatively, it may be possible to produce the protein in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Potentiallysuitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida albicans, orany yeast strain capable of expressing heterologous proteins.Potentially suitable bacterial strains include Escherichia coli,Bacillus subtilis, Salmonella typhimurium, or any bacterial straincapable of expressing heterologous proteins. If the protein is made inyeast or bacteria, it may be necessary to modify the protein producedtherein, for example by phosphorylation or glycosylation of theappropriate sites, in order to obtain the functional protein. Suchcovalent attachments may be accomplished using known chemical orenzymatic methods.

In another embodiment of the present invention, cells and tissues may beengineered to express an endogenous gene comprising the polynucleotidesof the invention under the control of inducible regulatory elements, inwhich case the regulatory sequences of the endogenous gene may bereplaced by homologous recombination. As described herein, genetargeting can be used to replace a gene's existing regulatory regionwith a regulatory sequence isolated from a different gene or a novelregulatory sequence synthesized by genetic engineering methods. Suchregulatory sequences may be comprised of promoters, enhancers,scaffold-attachment regions, negative regulatory elements,transcriptional initiation sites, regulatory protein binding sites orcombinations of said sequences. Alternatively, sequences which affectthe structure or stability of the RNA or protein produced may bereplaced, removed, added, or otherwise modified by targeting, includingpolyadenylation signals, mRNA stability elements, splice sites, leadersequences for enhancing or modifying transport or secretion propertiesof the protein, or other sequences which alter or improve the functionor stability of protein or RNA molecules.

The targeting event may be a simple insertion of the regulatorysequence, placing the gene under the control of the new regulatorysequence, e.g., inserting a new promoter or enhancer or both upstream ofa gene. Alternatively, the targeting event may be a simple deletion of aregulatory element, such as the deletion of a tissue-specific negativeregulatory element. Alternatively, the targeting event may replace anexisting element; for example, a tissue-specific enhancer can bereplaced by an enhancer that has broader or different cell-typespecificity than the naturally occurring elements. Here, the naturallyoccurring sequences are deleted and new sequences are added. In allcases, the identification of the targeting event may be facilitated bythe use of one or more selectable marker genes that are contiguous withthe targeting DNA, allowing for the selection of cells in which theexogenous DNA has integrated into the host cell genome. Theidentification of the targeting event may also be facilitated by the useof one or more marker genes exhibiting the property of negativeselection, such that the negatively selectable marker is linked to theexogenous DNA, but configured such that the negatively selectable markerflanks the targeting sequence, and such that a correct homologousrecombination event with sequences in the host cell genome does notresult in the stable integration of the negatively selectable marker.Markers useful for this purpose include the Herpes Simplex Virusthymidine kinase (TK) gene or the bacterial xanthine-guaninephosphoribosyl-transferase (gpt) gene.

The gene targeting or gene activation techniques which can be used inaccordance with this aspect of the invention are more particularlydescribed in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461to Sherwin et al.; International Application No. PCT/US92/09627(WO93/09222) by Selden et al.; and International Application No.PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which isincorporated by reference herein in its entirety.

4.13.1 Chimeric and Fusion Proteins

The invention also provides chimeric or fusion proteins. As used herein,a “chimeric protein” or “fusion protein” of the invention comprises apolypeptide of the invention operatively linked to another polypeptide.Within a fusion protein of the invention, the polypeptide according tothe invention can correspond to all or a portion of a protein accordingto the invention. In one embodiment, a fusion protein comprises at leastone biologically active portion of a protein according to the invention.In another embodiment, a fusion protein comprises at least twobiologically active portions of a protein according to the invention. Inyet another embodiment, a fusion protein comprises at least threebiologically active portions of a protein according to the invention.Within the fusion protein, the term “operatively-linked” is intended toindicate that the polypeptide according to the invention and the otherpolypeptide are fused in-frame with one another. The other polypeptidecan be fused to the N-terminus or C-terminus of the polypeptideaccording to the invention. For example, in one embodiment a fusionprotein comprises a polypeptide according to the invention operablylinked to the extracellular domain of a second protein.

In one embodiment, the fusion protein is a GST-fusion protein in whichthe polypeptide sequences according to the invention are fused to theC-terminus of the GST (glutathione S-transferase) sequences. Such fusionproteins can facilitate the purification of recombinant polypeptidesaccording to the invention. In another embodiment, the fusion protein isa protein according to the invention containing a heterologous signalsequence at its N-terminus. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of the polypeptide according to theinvention can be increased through use of a heterologous signalsequence.

In yet another embodiment, the fusion protein is an immunoglobulinfusion protein in which the polypeptide sequences of the invention arefused to sequences derived from a member of the immunoglobulin proteinfamily. The immunoglobulin fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a ligand and a proteinaccording to the invention on the surface of a cell, to thereby suppresssignal transduction mediated by the protein according to the inventionin vivo. The immunoglobulin fusion proteins can be used to affect thebioavailability of a cognate ligand. Inhibition of the ligand/proteininteraction can be useful therapeutically for both the treatment ofproliferative and differentiative disorders, as well as modulating (e.g.promoting or inhibiting) cell survival. Moreover, the immunoglobulinfusion proteins of the invention can be used as immunogens to produceantibodies in a subject, to purify ligands, and in screening assays toidentify molecules that inhibit the interaction of a polypeptideaccording to the invention with a ligand.

A chimeric or fusion protein of the invention can be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons, 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A nucleic acid encoding a polypeptide of the invention canbe cloned into such an expression vector such that the fusion moiety islinked in-frame to the protein of the invention.

4.14 Polypeptides of the Invention

The isolated polypeptides of the invention include, but are not limitedto, a polypeptide comprising: the amino acid sequence set forth as anyone of SEQ ID NO: 5, 7-13, 15, 17-24,28,30-156, 160, 162-182, 186,188-213, 215, 217-239, 241, 243-270, 272, 274-299, 302, 304-321, 323,325-344, 348, 350-352, 355, 357-376, 378, 380-401, 408, 410-414, 415,420, 422-439, 444-480, 482-484, 487, 489-501, 505, 507-512, 516,518-524, 528, 530-539, 542, 544-546, 548, 550-553, 557, 559-567, 572,574, 576, 579, 581-584, 588, 590, 596, 602, 604-605, 607, 609-610, 612,614-615, 618, 620, 622, 624, 626, 628, 630, 632, or 634-653 or an aminoacid sequence encoded by any one of the nucleotide sequences SEQ ID NO:2-4, 6, 14, 16, 26-27, 29, 158-159, 161, 184-185, 187, 214, 216, 240,242, 271, 273, 301, 303, 322, 324, 346-347, 349, 354, 356, 377, 379,407, 409, 419, 421, 443, 486, 488, 504, 506, 515, 517, 527, 529, 541,543, 547, 549, 556, 558, 571, 573, 578, 580, 587, 589, 601, 603, 606,608, 611, 613, 617, 619, 621, 623, 625, 627, 630, or 631, or thecorresponding full length or mature protein. Polypeptides of theinvention also include polypeptides preferably with biological orimmunological activity that are encoded by: (a) a polynucleotide havingany one of the nucleotide sequences set forth in SEQ ID NO: 1-4, 6, 14,16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273,300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407,409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517,526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578,580,587,589,601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631or (b) polynucleotides encoding any one of the amino acid sequences setforth as SEQ ID NO: 5, 7-13, 15, 17-24, 28, 30-156, 160, 162-182, 186,188-213, 215, 217-239, 241, 243-270, 272, 274-299, 302, 304-321, 323,325-344, 348, 350-352, 355, 357-376, 378, 380-401, 408, 410-414, 415,420, 422-439, 444-480, 482-484, 487, 489-501, 505, 507-512, 516,518-524, 528, 530-539, 542, 544-546, 548, 550-553, 557, 559-567, 572,574, 576, 579, 581-584, 588, 590, 596, 602, 604-605, 607, 609-610, 612,614-615, 618, 620, 622, 624, 626, 628, 630, 632, or 634-653 or (c)polynucleotides that hybridize to the complement of the polynucleotidesof either (a) or (b) under stringent hybridization conditions. Theinvention also provides biologically active or immunologically activevariants of any of the amino acid sequences set forth as SEQ ID NO: 5,7-13, 15, 17-24, 28, 30-156, 160, 162-182, 186, 188-213, 215, 217-239,241, 243-270, 272, 274-299, 302, 304-321, 323, 325-344, 348, 350-352,355, 357-376, 378, 380-401, 408, 410-414, 415, 420, 422-439, 444-480,482-484, 487, 489-501, 505, 507-512, 516, 518-524, 528, 530-539, 542,544-546, 548, 550-553, 557, 559-567, 572, 574, 576, 579,581-584,588,590,596, 602, 604-605,607,609-610,612,614-615, 618, 620,622, 624, 626, 628, 630, 632, or 634-653 or the corresponding fulllength or mature protein; and “substantial equivalents” thereof (e.g.,with at least about 65%, at least about 70%, at least about 75%, atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, moretypically at least about 90%, 91%, 92%, 93%, or 94% and even moretypically at least about 95%, 96%, 97%, 98% or 99%, most typically atleast about 99% amino acid identity) that retain biological activity.Polypeptides encoded by allelic variants may have a similar, increased,or decreased activity compared to polypeptides comprising SEQ ID NO: 5,7-13, 15, 17-24, 28, 30-156, 160, 162-182, 186, 188-213, 215, 217-239,241, 243-270, 272, 274-299, 302, 304-321, 323, 325-344, 348, 350-352,355, 357-376, 378, 380-401, 408, 410-414, 415, 420, 422-439, 444-480,482-484, 487, 489-501, 505, 507-512, 516, 518-524, 528, 530-539, 542,544-546, 548, 550-553, 557, 559-567, 572, 574, 576, 579, 581-584, 588,590, 596, 602, 604-605, 607, 609-610, 612, 614-615, 618, 620, 622, 624,626, 628, 630, 632, or 634-653.

Fragments of the proteins of the present invention which are capable ofexhibiting biological activity are also encompassed by the presentinvention. Fragments of the protein may be in linear form or they may becyclized using known methods, for example, as described in H. U.Saragovi, et al., Bio/Technology 10:773-778 (1992) and in R. S.McDowell, et al., J. Amer. Chem. Soc. 114:9245-9253 (1992), both ofwhich are incorporated herein by reference. Such fragments may be fusedto carrier molecules such as immunoglobulins for many purposes,including increasing the valency of protein binding sites.

The present invention also provides both full-length and mature forms(for example, without a signal sequence or precursor sequence) of thedisclosed proteins. The protein coding sequence is identified in thesequence listing by translation of the disclosed nucleotide sequences.The mature form of such protein may be obtained by expression of afull-length polynucleotide in a suitable mammalian cell or other hostcell. The sequence of the mature form of the protein is alsodeterminable from the amino acid sequence of the full-length form. Whereproteins of the present invention are membrane bound, soluble forms ofthe proteins are also provided. In such forms, part or all of theregions causing the proteins to be membrane bound are deleted so thatthe proteins are fully secreted from the cell in which it is expressed.

Protein compositions of the present invention may further comprise anacceptable carrier, such as a hydrophilic, e.g., pharmaceuticallyacceptable, carrier.

The present invention further provides isolated polypeptides encoded bythe nucleic acid fragments of the present invention or by degeneratevariants of the nucleic acid fragments of the present invention. By“degenerate variant” is intended nucleotide fragments which differ froma nucleic acid fragment of the present invention (e.g., an ORF) bynucleotide sequence but, due to the degeneracy of the genetic code,encode an identical polypeptide sequence. Preferred nucleic acidfragments of the present invention are the ORFs that encode proteins.

A variety of methodologies known in the art can be utilized to obtainany one of the isolated polypeptides or proteins of the presentinvention. At the simplest level, the amino acid sequence can besynthesized using commercially available peptide synthesizers. Thesynthetically-constructed protein sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with proteins may possess biological properties incommon therewith, including protein activity. This technique isparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the native polypeptide. Thus, they may be employed asbiologically active or immunological substitutes for natural, purifiedproteins in screening of therapeutic compounds and in immunologicalprocesses for the development of antibodies.

The polypeptides and proteins of the present invention can alternativelybe purified from cells which have been altered to express the desiredpolypeptide or protein. As used herein, a cell is said to be altered toexpress a desired polypeptide or protein when the cell, through geneticmanipulation, is made to produce a polypeptide or protein which itnormally does not produce or which the cell normally produces at a lowerlevel. One skilled in the art can readily adapt procedures forintroducing and expressing either recombinant or synthetic sequencesinto eukaryotic or prokaryotic cells in order to generate a cell whichproduces one of the polypeptides or proteins of the present invention.

The invention also relates to methods for producing a polypeptidecomprising growing a culture of host cells of the invention in asuitable culture medium, and purifying the protein from the cells or theculture in which the cells are grown. For example, the methods of theinvention include a process for producing a polypeptide in which a hostcell containing a suitable expression vector that includes apolynucleotide of the invention is cultured under conditions that allowexpression of the encoded polypeptide. The polypeptide can be recoveredfrom the culture, conveniently from the culture medium, or from a lysateprepared from the host cells and further purified. Preferred embodimentsinclude those in which the protein produced by such process is a fulllength or mature form of the protein.

In an alternative method, the polypeptide or protein is purified frombacterial cells which naturally produce the polypeptide or protein. Oneskilled in the art can readily follow known methods for isolatingpolypeptides and proteins in order to obtain one of the isolatedpolypeptides or proteins of the present invention. These include, butare not limited to, immunochromatography, HPLC, size-exclusionchromatography, ion-exchange chromatography, and immuno-affinitychromatography. See, e.g., Scopes, Protein Purification: Principles andPractice, Springer-Verlag (1994); Sambrook, et al., in MolecularCloning: A Laboratory Manual; Ausubel et al., Current Protocols inMolecular Biology. Polypeptide fragments that retainbiological/immunological activity include fragments comprising greaterthan about 100 amino acids, or greater than about 200 amino acids, andfragments that encode specific protein domains.

The purified polypeptides can be used in in vitro binding assays whichare well known in the art to identify molecules which bind to thepolypeptides. These molecules include but are not limited to, for e.g.,small molecules, molecules from combinatorial libraries, antibodies orother proteins. The molecules identified in the binding assay are thentested for antagonist or agonist activity in in vivo tissue culture oranimal models that are well known in the art. In brief, the moleculesare titrated into a plurality of cell cultures or animals and thentested for either cell/animal death or prolonged survival of theanimal/cells.

In addition, the peptides of the invention or molecules capable ofbinding to the peptides may be complexed with toxins, e.g., ricin orcholera, or with other compounds that are toxic to cells. Thetoxin-binding molecule complex is then targeted to a tumor or other cellby the specificity of the binding molecule for SEQ ID NO: 5, 7-13, 15,17-24, 28, 30-156, 160, 162-182, 186, 188-213, 215, 217-239, 241,243-270, 272, 274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355,357-376, 378, 380401, 408, 410-414, 415, 420, 422-439, 444-480, 482-484,487, 489-501, 505, 507-512, 516, 518-524, 528, 530-539, 542, 544-546,548, 550-553, 557, 559-567, 572, 574, 576, 579, 581-584, 588, 590, 596,602, 604-605, 607, 609-610, 612, 614-615, 618, 620, 622, 624, 626, 628,630, 632, or 634-653.

The protein of the invention may also be expressed as a product oftransgenic animals, e.g., as a component of the milk of transgenic cows,goats, pigs, or sheep which are characterized by somatic or germ cellscontaining a nucleotide sequence encoding the protein.

The proteins provided herein also include proteins characterized byamino acid sequences similar to those of purified proteins but intowhich modification are naturally provided or deliberately engineered.For example, modifications, in the peptide or DNA sequence, can be madeby those skilled in the art using known techniques. Modifications ofinterest in the protein sequences may include the alteration,substitution, replacement, insertion or deletion of a selected aminoacid residue in the coding sequence. For example, one or more of thecysteine residues may be deleted or replaced with another amino acid toalter the conformation of the molecule. Techniques for such alteration,substitution, replacement, insertion or deletion are well known to thoseskilled in the art (see, e.g. U.S. Pat. No. 4,518,584). Preferably, suchalteration, substitution, replacement, insertion or deletion retains thedesired activity of the protein. Regions of the protein that areimportant for the protein function can be determined by various methodsknown in the art including the alanine-scanning method which involvedsystematic substitution of single or strings of amino acids withalanine, followed by testing the resulting alanine-containing variantfor biological activity. This type of analysis determines the importanceof the substituted amino acid(s) in biological activity. Regions of theprotein that are important for protein function may be determined by theeMATRIX program.

Other fragments and derivatives of the sequences of proteins which wouldbe expected to retain protein activity in whole or in part and areuseful for screening or other immunological methodologies may also beeasily made by those skilled in the art given the disclosures herein.Such modifications are encompassed by the present invention.

The protein may also be produced by operably linking the isolatedpolynucleotide of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form from, e.g., Invitrogen,San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are wellknown in the art, as described in Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987), incorporated herein byreference. As used herein, an insect cell capable of expressing apolynucleotide of the present invention is “transformed.”

The protein of the invention may be prepared by culturing transformedhost cells under culture conditions suitable to express the recombinantprotein. The resulting expressed protein may then be purified from suchculture (i.e., from culture medium or cell extracts) using knownpurification processes, such as gel filtration and ion exchangechromatography. Purification of the protein of the invention may alsoinclude an affinity column containing agents which will bind to theprotein of the invention; one or more column steps over such affinityresins as concanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue3GA Sepharose™; one or more steps involving hydrophobic interactionchromatography using such resins as phenyl ether, butyl ether, or propylether; or immunoaffinity chromatography.

Alternatively, the protein of the invention may also be expressed in aform which will facilitate purification. For example, it may beexpressed as a fusion protein, such as those of maltose binding protein(MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a Histag. Kits for expression and purification of such fusion proteins arecommercially available from New England BioLab (Beverly, Mass.),Pharmacia (Piscataway, N.J.) and Invitrogen, respectively. The proteinof the invention can also be tagged with an epitope and subsequentlypurified by using a specific antibody directed to such epitope. One suchepitope (“FLAG®”) is commercially available from Kodak (New Haven,Conn.).

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the protein of the invention. Some or all ofthe foregoing purification steps, in various combinations, can also beemployed to provide a substantially homogeneous isolated recombinantprotein. The protein thus purified is substantially free of othermammalian proteins and is defined in accordance with the presentinvention as an “isolated protein.”

The polypeptides of the invention include analogs (variants). Thisembraces fragments of the polypeptides of the invention, as wellpolypeptides of the invention which comprise one or more amino acidsdeleted, inserted, or substituted. Also, analogs of the polypeptides ofthe invention embrace fusions of the polypeptides of the invention ormodifications of the polypeptides of the invention, wherein thepolypeptide or analog of the invention is fused to another moiety ormoieties, e.g., targeting moiety or another therapeutic agent. Suchanalogs may exhibit improved properties such as activity and/orstability. Examples of moieties which may be fused to the polypeptide oran analog of the invention include, for example, targeting moietieswhich provide for the delivery of polypeptides of the invention toneurons, e.g., antibodies to central nervous system, or antibodies toreceptor and ligands expressed on neuronal cells. Other moieties whichmay be fused to polypeptides of the invention include therapeutic agentswhich are used for treatment, for example antidepressant drugs or othermedications for neurological disorders. Also, polypeptides of theinvention may be fused to neuron growth modulators, and other chemokinesfor targeted delivery.

4.14.1 Determining Polypeptide and Polynucleotide Identity andSimilarity

Preferred identity and/or similarity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in computer programs including, but are notlimited to, the GCG program package, including GAP (Devereux, J., etal., Nucl. Acids Res. 12:387 (1984); Genetics Computer Group, Universityof Wisconsin, Madison, Wis., herein incorporated by reference), BLASTP,BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. Molec. Biol.215:403-410 (1990), PSI-BLAST (Altschul S. F. et al., Nucl. Acids Res.25:3389-3402, herein incorporated by reference), the eMatrix software(Wu et al., J. Comp. Biol., 6:219-235 (1999), herein incorporated byreference), eMotif software (Nevill-Manning et al, ISMB-97, 4:202-209,herein incorporated by reference), the GeneAtlas software (MolecularSimulations Inc. (MSI), San Diego, Calif.) (Sanchez and Sali, Proc.Natl. Acad. Sci. USA, 95:13597-13602 (1998); Kitson D H, et al, (2000)“Remote homology detection using structural modeling—an evaluation”Submitted; Fischer and Eisenberg, Protein Sci. 5:947-955 (1996)), andthe Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol,157:105-31 (1982), incorporated herein by reference). The BLAST programsare publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul, S., et al.NCB NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol.215:403-410 (1990).

4.15 Gene Therapy

Mutations in the gene encoding the polypeptide of the invention mayresult in loss of normal function of the encoded protein. The inventionthus provides gene therapy to restore normal activity of thepolypeptides of the invention; or to treat disease states involvingpolypeptides of the invention. Delivery of a functional gene encodingpolypeptides of the invention to appropriate cells is effected ex vivo,in situ, or in vivo by use of vectors, and more particularly viralvectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), orex vivo by use of physical DNA transfer methods (e.g., liposomes orchemical treatments). See, for example, Anderson, Nature,392(Suppl.):25-20 (1998). For additional reviews of gene therapytechnology see Friedmann, Science, 244:1275-1281 (1989); Verma,Scientific American: 68-84 (1990); and Miller, Nature, 357:455-460(1992). Introduction of any one of the nucleotides of the presentinvention or a gene encoding the polypeptides of the present inventioncan also be accomplished with extrachromosomal substrates (transientexpression) or artificial chromosomes (stable expression). Cells mayalso be cultured ex vivo in the presence of proteins of the presentinvention in order to proliferate or to produce a desired effect on oractivity in such cells. Treated cells can then be introduced in vivo fortherapeutic purposes. Alternatively, it is contemplated that in otherhuman disease states, preventing the expression of or inhibiting theactivity of polypeptides of the invention will be useful in treating thedisease states. It is contemplated that antisense therapy or genetherapy could be applied to negatively regulate the expression ofpolypeptides of the invention.

Other methods inhibiting expression of a protein include theintroduction of antisense molecules to the nucleic acids of the presentinvention, their complements, or their translated RNA sequences, bymethods known in the art. Further, the polypeptides of the presentinvention can be inhibited by using targeted deletion methods, or theinsertion of a negative regulatory element such as a silencer, which istissue specific.

The present invention still further provides cells geneticallyengineered in vivo to express the polynucleotides of the invention,wherein such polynucleotides are in operative association with aregulatory sequence heterologous to the host cell which drivesexpression of the polynucleotides in the cell. These methods can be usedto increase or decrease the expression of the polynucleotides of thepresent invention.

Knowledge of DNA sequences provided by the invention allows formodification of cells to permit, increase, or decrease, expression ofendogenous polypeptide. Cells can be modified (e.g., by homologousrecombination) to provide increased polypeptide expression by replacing,in whole or in part, the naturally occurring promoter with all or partof a heterologous promoter so that the cells express the protein athigher levels. The heterologous promoter is inserted in such a mannerthat it is operatively linked to the desired protein encoding sequences.See, for example, PCT International Publication No. WO 94/12650, PCTInternational Publication No. WO 92/20808, and PCT InternationalPublication No. WO 91/09955. It is also contemplated that, in additionto heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr,and the multifunctional CAD gene which encodes carbamyl phosphatesynthase, aspartate transcarbamylase, and dihydroorotase) and/or intronDNA may be inserted along with the heterologous promoter DNA. If linkedto the desired protein coding sequence, amplification of the marker DNAby standard selection methods results in co-amplification of the desiredprotein coding sequences in the cells.

In another embodiment of the present invention, cells and tissues may beengineered to express an endogenous gene comprising the polynucleotidesof the invention under the control of inducible regulatory elements, inwhich case the regulatory sequences of the endogenous gene may bereplaced by homologous recombination. As described herein, genetargeting can be used to replace a gene's existing regulatory regionwith a regulatory sequence isolated from a different gene or a novelregulatory sequence synthesized by genetic engineering methods. Suchregulatory sequences may be comprised of promoters, enhancers,scaffold-attachment regions, negative regulatory elements,transcriptional initiation sites, regulatory protein binding sites orcombinations of said sequences. Alternatively, sequences which affectthe structure or stability of the RNA or protein produced may bereplaced, removed, added, or otherwise modified by targeting. Thesesequences include polyadenylation signals, mRNA stability elements,splice sites, leader sequences for enhancing or modifying transport orsecretion properties of the protein, or other sequences which alter orimprove the function or stability of protein or RNA molecules.

The targeting event may be a simple insertion of the regulatorysequence, placing the gene under the control of the new regulatorysequence, e.g., inserting a new promoter or enhancer or both upstream ofa gene. Alternatively, the targeting event may be a simple deletion of aregulatory element, such as the deletion of a tissue-specific negativeregulatory element. Alternatively, the targeting event may replace anexisting element; for example, a tissue-specific enhancer can bereplaced by an enhancer that has broader or different cell-typespecificity than the naturally occurring elements. Here, the naturallyoccurring sequences are deleted and new sequences are added. In allcases, the identification of the targeting event may be facilitated bythe use of one or more selectable marker genes that are contiguous withthe targeting DNA, allowing for the selection of cells in which theexogenous DNA has integrated into the cell genome. The identification ofthe targeting event may also be facilitated by the use of one or moremarker genes exhibiting the property of negative selection, such thatthe negatively selectable marker is linked to the exogenous DNA, butconfigured such that the negatively selectable marker flanks thetargeting sequence, and such that a correct homologous recombinationevent with sequences in the host cell genome does not result in thestable integration of the negatively selectable marker. Markers usefulfor this purpose include the Herpes Simplex Virus thymidine kinase (TK)gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt)gene.

The gene targeting or gene activation techniques which can be used inaccordance with this aspect of the invention are more particularlydescribed in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461to Sherwin et al.; International Application No. PCT/US92/09627(WO93/09222) by Selden et al.; and International Application No.PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which isincorporated by reference herein in its entirety.

4.16 Transgenic Animals

In preferred methods to determine biological functions of thepolypeptides of the invention in vivo, one or more genes provided by theinvention are either over expressed or inactivated in the germ line ofanimals using homologous recombination (Capecchi, Science 244:1288-1292(1989)). Animals in which the gene is over expressed, under theregulatory control of exogenous or endogenous promoter elements, areknown as transgenic animals. Animals in which an endogenous gene hasbeen inactivated by homologous recombination are referred to as“knockout” animals. Knockout animals, preferably non-human mammals, canbe prepared as described in U.S. Pat. No. 5,557,032, incorporated hereinby reference. Transgenic animals are useful to determine the rolespolypeptides of the invention play in biological processes, andpreferably in disease states. Transgenic animals are useful as modelsystems to identify compounds that modulate lipid metabolism. Transgenicanimals, preferably non-human mammals, are produced using methods asdescribed in U.S. Pat. No. 5,489,743 and PCT Publication No. WO94/28122,incorporated herein by reference.

Transgenic animals can be prepared wherein all or part of a promoter ofthe polynucleotides of the invention is either activated or inactivatedto alter the level of expression of the polypeptides of the invention.Inactivation can be carried out using homologous recombination methodsdescribed above. Activation can be achieved by supplementing or evenreplacing the homologous promoter to provide for increased proteinexpression. The homologous promoter can be supplemented by insertion ofone or more heterologous enhancer elements known to confer promoteractivation in a particular tissue.

The polynucleotides of the present invention also make possible thedevelopment, through, e.g., homologous recombination or knock outstrategies, of animals that fail to express functional polypeptides ofthe invention or that express a variant of the polypeptides of theinvention. Such animals are useful as models for studying the in vivoactivities of polypeptides of the invention as well as for studyingmodulators of the polypeptides of the invention.

4.17 Uses and Biological Activity

The polynucleotides and proteins of the present invention are expectedto exhibit one or more of the uses or biological activities (includingthose associated with assays cited herein) identified herein. Uses oractivities described for proteins of the present invention may beprovided by administration or use of such proteins or of polynucleotidesencoding such proteins (such as, for example, in gene therapies orvectors suitable for introduction of DNA). The mechanism underlying theparticular condition or pathology will dictate whether the polypeptidesof the invention, the polynucleotides of the invention or modulators(activators or inhibitors) thereof would be beneficial to the subject inneed of treatment. Thus, “therapeutic compositions of the invention”include compositions comprising isolated polynucleotides (includingrecombinant DNA molecules, cloned genes and degenerate variants thereof)or polypeptides of the invention (including full length protein, matureprotein and truncations or domains thereof), or compounds and othersubstances that modulate the overall activity of the target geneproducts, either at the level of target gene/protein expression ortarget protein activity. Such modulators include polypeptides, analogs,(variants), including fragments and fusion proteins, antibodies andother binding proteins; chemical compounds that directly or indirectlyactivate or inhibit the polypeptides of the invention (identified, e.g.,via drug screening assays as described herein); antisensepolynucleotides and polynucleotides suitable for triple helix formation;and in particular antibodies or other binding partners that specificallyrecognize one or more epitopes of the polypeptides of the invention.

The polypeptides of the present invention may likewise be involved incellular activation or in one of the other physiological pathwaysdescribed herein.

4.17.1 Research Uses and Utilities

The polynucleotides provided by the present invention can be used by theresearch community for various purposes. The polynucleotides can be usedto express recombinant protein for analysis, characterization ortherapeutic use; as markers for tissues in which the correspondingprotein is preferentially expressed (either constitutively or at aparticular stage of tissue differentiation or development or in diseasestates); as molecular weight markers on gels; as chromosome markers ortags (when labeled) to identify chromosomes or to map related genepositions; to compare with endogenous DNA sequences in patients toidentify potential genetic disorders; as probes to hybridize and thusdiscover novel, related DNA sequences; as a source of information toderive PCR primers for genetic fingerprinting; as a probe to“subtract-out” known sequences in the process of discovering other novelpolynucleotides; for selecting and making oligomers for attachment to a“gene chip” or other support, including for examination of expressionpatterns; to raise anti-protein antibodies using DNA immunizationtechniques; and as an antigen to raise anti-DNA antibodies or elicitanother immune response. Where the polynucleotide encodes a proteinwhich binds or potentially binds to another protein (such as, forexample, in a receptor-ligand interaction), the polynucleotide can alsobe used in interaction trap assays (such as, for example, that describedin Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotidesencoding the other protein with which binding occurs or to identifyinhibitors of the binding interaction.

The polypeptides provided by the present invention can similarly be usedin assays to determine biological activity, including in a panel ofmultiple proteins for high-throughput screening; to raise antibodies orto elicit another immune response; as a reagent (including the labeledreagent) in assays designed to quantitatively determine levels of theprotein (or its receptor) in biological fluids; as markers for tissuesin which the corresponding polypeptide is preferentially expressed(either constitutively or at a particular stage of tissuedifferentiation or development or in a disease state); and, of course,to isolate correlative receptors or ligands. Proteins involved in thesebinding interactions can also be used to screen for peptide or smallmolecule inhibitors or agonists of the binding interaction.

The polypeptides of the invention are also useful for making antibodysubstances that are specifically immunoreactive with proteins accordingto the invention. Antibodies and portions thereof (e.g., Fab fragments)which bind to the polypeptides of the invention can be used to identifythe presence of such polypeptides in a sample. Such determinations arecarried out using any suitable immunoassay format, and any polypeptideof the invention that is specifically bound by the antibody can beemployed as a positive control.

Any or all of these research utilities are capable of being developedinto reagent grade or kit format for commercialization as researchproducts.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include withoutlimitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold SpringHarbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatiseds., 1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

4.17.2 Cytokine and Cell Proliferation/Differentiation Activity

A polypeptide of the present invention may exhibit activity relating tocytokine, cell proliferation (either inducing or inhibiting) or celldifferentiation (either inducing or inhibiting) activity or may induceproduction of other cytokines in certain cell populations. Apolynucleotide of the invention can encode a polypeptide exhibiting suchattributes. Many protein factors discovered to date, including all knowncytokines, have exhibited activity in one or more factor-dependent cellproliferation assays, and hence the assays serve as a convenientconfirmation of cytokine activity. The activity of therapeuticcompositions of the present invention is evidenced by any one of anumber of routine factor dependent cell proliferation assays for celllines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1,Mo7e, CMK, HUVEC, and Caco. Therapeutic compositions of the inventioncan be used in the following:

Assays for T-cell or thymocyte proliferation include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, InVitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,Immunologic studies in Humans); Takai, et al., J. Immunol. 137:3494-3500(1986); Bertagnolli, et al., J. Immunol. 145:1706-1712 (1990);Bertagnolli, et al., Cellular Immunology 133:327-341 (1991);Bertagnolli, et al., J. Immunol. 149:3778-3783 (1992); Bowman, et al.,J. Immunol. 152:1756-1761 (1994).

Assays for cytokine production and/or proliferation of spleen cells,lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation, Kruisbeek, A. M. andShevach, E. M. In Current Protocols in Immunology. J. E. e.a. Coliganeds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; andMeasurement of mouse and human interferon-γ, Schreiber, R. D. In CurrentProtocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8,John Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Measurement of Human and Murine Interleukin 2 and Interleukin 4,Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols inImmunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wileyand Sons, Toronto. 1991; deVries, et al., J. Exp. Med. 173:1205-1211(1991); Moreau, et al., Nature 336:690-692 (1988); Greenberger, et al.,Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938 (1983); Measurement of mouseand human interleukin 6—Nordan, R. In Current Protocols in Immunology.J. E. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto.1991; Smith, et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861 (1986);Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark,S. C. and Turner, K. J. In Current Protocols in Immunology. J. E.Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;Measurement of mouse and human Interleukin 9-Ciarletta, A., Giannotti,J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology.J. E. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.

Assays for T-cell clone responses to antigens (which will identify,among others, proteins that affect APC-T cell interactions as well asdirect T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction; Chapter 6, Cytokines and their cellular receptors; Chapter 7,Immunologic studies in Humans); Weinberger, et al., Proc. Natl. Acad.Sci. USA 77:6091-6095 (1980); Weinberger, et al., Eur. J. Immun.11:405-411 (1981); Takai, et al., J. Immunol. 137:3494-3500 (1986);Takai, et al., J. Immunol. 140:508-512 (1988).

4.17.3 Stem Cell Growth Factor Activity

A polypeptide of the present invention may exhibit stem cell growthfactor activity and be involved in the proliferation, differentiationand survival of pluripotent and totipotent stem cells includingprimordial germ cells, embryonic stem cells, hematopoietic stem cellsand/or germ line stem cells. Administration of the polypeptide of theinvention to stem cells in vivo or ex vivo may maintain and expand cellpopulations in a totipotential or pluripotential state which would beuseful for re-engineering damaged or diseased tissues, transplantation,and manufacture of bio-pharmaceuticals and the development ofbio-sensors. The ability to produce large quantities of human cells hasimportant working applications for the production of human proteinswhich currently must be obtained from non-human sources or donors,implantation of cells to treat diseases such as Parkinson's, Alzheimer'sand other neurodegenerative diseases; tissues for grafting such as bonemarrow, skin, cartilage, tendons, bone, muscle (including cardiacmuscle), blood vessels, cornea, neural cells, gastrointestinal cells andothers; and organs for transplantation such as kidney, liver, pancreas(including islet cells), heart and lung.

It is contemplated that multiple different exogenous growth factorsand/or cytokines may be administered in combination with the polypeptideof the invention to achieve the desired effect, including any of thegrowth factors listed herein, other stem cell maintenance factors, andspecifically including stem cell factor (SCF), leukemia inhibitoryfactor (LIF), Flt-3 ligand (Flt-3L), any of the interleukins,recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatoryprotein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO),platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), neuralgrowth factors and basic fibroblast growth factor (bFGF).

Since totipotent stem cells can give rise to virtually any mature celltype, expansion of these cells in culture will facilitate the productionof large quantities of mature cells. Techniques for culturing stem cellsare known in the art and administration of polypeptides of theinvention, optionally with other growth factors and/or cytokines, isexpected to enhance the survival and proliferation of the stem cellpopulations. This can be accomplished by direct administration of thepolypeptide of the invention to the culture medium. Alternatively,stroma cells transfected with a polynucleotide that encodes for thepolypeptide of the invention can be used as a feeder layer for the stemcell populations in culture or in vivo. Stromal support cells for feederlayers may include embryonic bone marrow fibroblasts, bone marrowstromal cells, fetal liver cells, or cultured embryonic fibroblasts (seeU.S. Pat. No. 5,690,926).

Stem cells themselves can be transfected with a polynucleotide of theinvention to induce autocrine expression of the polypeptide of theinvention. This will allow for generation of undifferentiatedtotipotential/pluripotential stem cell lines that are useful as is orthat can then be differentiated into the desired mature cell types.These stable cell lines can also serve as a source of undifferentiatedtotipotential/pluripotential mRNA to create cDNA libraries and templatesfor polymerase chain reaction experiments. These studies would allow forthe isolation and identification of differentially expressed genes instem cell populations that regulate stem cell proliferation and/ormaintenance.

Expansion and maintenance of totipotent stem cell populations will beuseful in the treatment of many pathological conditions. For example,polypeptides of the present invention may be used to manipulate stemcells in culture to give rise to neuroepithelial cells that can be usedto augment or replace cells damaged by illness, autoimmune disease,accidental damage or genetic disorders. The polypeptide of the inventionmay be useful for inducing the proliferation of neural cells and for theregeneration of nerve and brain tissue, i.e. for the treatment ofcentral and peripheral nervous system diseases and neuropathies, as wellas mechanical and traumatic disorders which involve degeneration, deathor trauma to neural cells or nerve tissue. Furthermore, these cells canbe cultured in vitro to form other differentiated cells, such as skintissue that can be used for transplantation. In addition, the expandedstem cell populations can also be genetically altered for gene therapypurposes and to decrease host rejection of replacement tissues aftergrafting or implantation.

Expression of the polypeptide of the invention and its effect on stemcells can also be manipulated to achieve controlled differentiation ofthe stem cells into more differentiated cell types. A broadly applicablemethod of obtaining pure populations of a specific differentiated celltype from undifferentiated stem cell populations involves the use of acell-type specific promoter driving a selectable marker. The selectablemarker allows only cells of the desired type to survive. For example,stem cells can be induced to differentiate into cardiomyocytes (Wobus etal., Differentiation, 48:173-182 (1991); Klug, et al., J. Clin. Invest.,98:216-224 (1998)) or skeletal muscle cells (Browder, L. W. In:Principles of Tissue Engineering eds. Lanza, et al., Academic Press(1997)). Alternatively, directed differentiation of stem cells can beaccomplished by culturing the stem cells in the presence of adifferentiation factor such as retinoic acid and an antagonist of thepolypeptide of the invention which would inhibit the effects ofendogenous stem cell factor activity and allow differentiation toproceed.

In vitro cultures of stem cells can be used to determine if thepolypeptide of the invention exhibits stem cell growth factor activity.Stem cells are isolated from any one of various cell sources (includinghematopoietic stem cells and embryonic stem cells) and cultured on afeeder layer, as described by Thompson, et al. Proc. Natl. Acad. Sci,U.S.A., 92:7844-7848 (1995), in the presence of the polypeptide of theinvention alone or in combination with other growth factors orcytokines. The ability of the polypeptide of the invention to inducestem cells proliferation is determined by colony formation on semi-solidsupport e.g. as described by Bernstein, et al., Blood, 77: 2316-2321(1991).

4.17.4 Hematopoiesis Regulating Activity

A polypeptide of the present invention may be involved in regulation ofhematopoiesis and, consequently, in the treatment of myeloid or lymphoidcell disorders. Even marginal biological activity in support of colonyforming cells or of factor-dependent cell lines indicates involvement inregulating hematopoiesis, e.g. in supporting the growth andproliferation of erythroid progenitor cells alone or in combination withother cytokines, thereby indicating utility, for example, in treatingvarious anemias or for use in conjunction with irradiation/chemotherapyto stimulate the production of erythroid precursors and/or erythroidcells; in supporting the growth and proliferation of myeloid cells suchas granulocytes and monocytes/macrophages (i.e., traditional colonystimulating factor activity) useful, for example, in conjunction withchemotherapy to prevent or treat consequent myelo-suppression; insupporting the growth and proliferation of megakaryocytes andconsequently of platelets thereby allowing prevention or treatment ofvarious platelet disorders such as thrombocytopenia, and generally foruse in place of or complimentary to platelet transfusions; and/or insupporting the growth and proliferation of hematopoietic stem cellswhich are capable of maturing to any and all of the above-mentionedhematopoietic cells and therefore find therapeutic utility in variousstem cell disorders (such as those usually treated with transplantation,including, without limitation, aplastic anemia and paroxysmal nocturnalhemoglobinuria), as well as in repopulating the stem cell compartmentpost irradiation/chemotherapy, either in vivo or ex vivo (i.e., inconjunction with bone marrow transplantation or with peripheralprogenitor cell transplantation (homologous or heterologous)) as normalcells or genetically manipulated for gene therapy.

Therapeutic compositions of the invention can be used in the following:

Suitable assays for proliferation and differentiation of varioushematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify,among others, proteins that influence embryonic differentiationhematopoiesis) include, without limitation, those described in:Johansson, et al. Cellular Biology 15:141-15 (1995); Keller, et al.,Mol. Cell. Biol. 13:473486 (1993); McClanahan, et al., Blood81:2903-2915 (1993).

Assays for stem cell survival and differentiation (which will identify,among others, proteins that regulate lympho-hematopoiesis) include,without limitation, those described in: Methylcellulose colony formingassays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.1994; Hirayama, et al., Proc. Natl. Acad. Sci. USA 89:5907-5911 (1992);Primitive hematopoietic colony forming cells with high proliferativepotential, McNiece, I. K. and Briddell, R. A. In Culture ofHematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,Wiley-Liss, Inc., New York, N.Y. 1994; Neben, et al., ExperimentalHematology 22:353-359 (1994); Cobblestone area forming cell assay,Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, etal. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long termbone marrow cultures in the presence of stromal cells, Spooncer, E.,Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y.1994; Long term culture initiating cell assay, Sutherland, H. J. InCulture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

4.17.5 Tissue Growth Activity

A polypeptide of the present invention also may be involved in bone,cartilage, tendon, ligament and/or nerve tissue growth or regeneration,as well as in wound healing and tissue repair and replacement, and inhealing of burns, incisions and ulcers.

A polypeptide of the present invention which induces cartilage and/orbone growth in circumstances where bone is not normally formed hasapplication in the healing of bone fractures and cartilage damage ordefects in humans and other animals. Compositions of a polypeptide,antibody, binding partner, or other modulator of the invention may haveprophylactic use in closed as well as open fracture reduction and alsoin the improved fixation of artificial joints. De novo bone formationinduced by an osteogenic agent contributes to the repair of congenital,trauma induced, or oncologic resection induced craniofacial defects, andalso is useful in cosmetic plastic surgery.

A polypeptide of this invention may also be involved in attractingbone-forming cells, stimulating growth of bone-forming cells, orinducing differentiation of progenitors of bone-forming cells. Treatmentof osteoporosis, osteoarthritis, bone degenerative disorders, orperiodontal disease, such as through stimulation of bone and/orcartilage repair or by blocking inflammation or processes of tissuedestruction (collagenase activity, osteoclast activity, etc.) mediatedby inflammatory processes may also be possible using the composition ofthe invention.

Another category of tissue regeneration activity that may involve thepolypeptide of the present invention is tendon/ligament formation.Induction of tendon/ligament-like tissue or other tissue formation incircumstances where such tissue is not normally formed has applicationin the healing of tendon or ligament tears, deformities and other tendonor ligament defects in humans and other animals. Such a preparationemploying a tendon/ligament-like tissue inducing protein may haveprophylactic use in preventing damage to tendon or ligament tissue, aswell as use in the improved fixation of tendon or ligament to bone orother tissues, and in repairing defects to tendon or ligament tissue. Denovo tendon/ligament-like tissue formation induced by a composition ofthe present invention contributes to the repair of congenital, traumainduced, or other tendon or ligament defects of other origin, and isalso useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions of the present invention mayprovide environment to attract tendon- or ligament-forming cells,stimulate growth of tendon- or ligament-forming cells, inducedifferentiation of progenitors of tendon- or ligament-forming cells, orinduce growth of tendon/ligament cells or progenitors ex vivo for returnin vivo to effect tissue repair. The compositions of the invention mayalso be useful in the treatment of tendinitis, carpal tunnel syndromeand other tendon or ligament defects. The compositions may also includean appropriate matrix and/or sequestering agent as a carrier as is wellknown in the art.

The compositions of the present invention may also be useful forproliferation of neural cells and for regeneration of nerve and braintissue, i.e. for the treatment of central and peripheral nervous systemdiseases and neuropathies, as well as mechanical and traumaticdisorders, which involve degeneration, death or trauma to neural cellsor nerve tissue. More specifically, a composition of the invention maybe used in the treatment of diseases of the peripheral nervous system,such as peripheral nerve injuries, peripheral neuropathy and localizedneuropathies, and central nervous system diseases, such as Alzheimer's,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome. Further conditions which may betreated in accordance with the present invention include mechanical andtraumatic disorders, such as spinal cord disorders, head trauma andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a composition of the invention.

Compositions of the invention may also be useful to promote better orfaster closure of non-healing wounds, including without limitationpressure ulcers, ulcers associated with vascular insufficiency, surgicaland traumatic wounds, and the like.

Compositions of the present invention may also be involved in thegeneration or regeneration of other tissues, such as organs (including,for example, pancreas, liver, intestine, kidney, skin, and endothelium),muscle (smooth, skeletal or cardiac) and vascular (including vascularendothelium) tissue, or for promoting the growth of cells comprisingsuch tissues. Part of the desired effects may be by inhibition ormodulation of fibrotic scarring may allow normal tissue to regenerate. Apolypeptide of the present invention may also exhibit angiogenicactivity.

A composition of the present invention may also be useful for gutprotection or regeneration and treatment of lung or liver fibrosis,reperfusion injury in various tissues, and conditions resulting fromsystemic cytokine damage.

A composition of the present invention may also be useful for promotingor inhibiting differentiation of tissues described above from precursortissues or cells; or for inhibiting the growth of tissues describedabove.

Therapeutic compositions of the invention can be used in the following:

Assays for tissue generation activity include, without limitation, thosedescribed in: International Patent Publication No. WO95/16035 (bone,cartilage, tendon); International Patent Publication No. WO95/05846(nerve, neuronal); International Patent Publication No. WO91/07491(skin, endothelium).

Assays for wound healing activity include, without limitation, thosedescribed in: Winter, Epidermal Wound Healing, pp. 71-112 (Maibach, H.I. and Rovee, D. T., eds.), Year Book Medical Publishers, Inc., Chicago,as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84(1978).

4.17.6 Immune Function Stimulating or Suppressing Activity

A polypeptide of the present invention may also exhibit immunestimulating or immune suppressing activity, including without limitationthe activities for which assays are described herein. A polynucleotideof the invention can encode a polypeptide exhibiting such activities. Aprotein may be useful in the treatment of various immune deficienciesand disorders (including severe combined immunodeficiency (SCID)), e.g.,in regulating (up or down) growth and proliferation of T and/or Blymphocytes, as well as effecting the cytolytic activity of NK cells andother cell populations. These immune deficiencies may be genetic or becaused by viral (e.g., HIV) as well as bacterial or fungal infections,or may result from autoimmune disorders. More specifically, infectiousdiseases causes by viral, bacterial, fungal or other infection may betreatable using a protein of the present invention, including infectionsby HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmaniaspp., malaria spp. and various fungal infections such as candidiasis. Ofcourse, in this regard, proteins of the present invention may also beuseful where a boost to the immune system generally may be desirable,i.e., in the treatment of cancer.

Autoimmune disorders which may be treated using a protein of the presentinvention include, for example, connective tissue disease, multiplesclerosis, systemic lupus erythematosus, rheumatoid arthritis,autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmunethyroiditis, insulin dependent diabetes mellitis, myasthenia gravis,graft-versus-host disease and autoimmune inflammatory eye disease. Sucha protein (or antagonists thereof, including antibodies) of the presentinvention may also to be useful in the treatment of allergic reactionsand conditions (e.g., anaphylaxis, serum sickness, drug reactions, foodallergies, insect venom allergies, mastocytosis, allergic rhinitis,hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopicdermatitis, allergic contact dermatitis, erythema multiforme,Stevens-Johnson syndrome, allergic conjunctivitis, atopickeratoconjunctivitis, venereal keratoconjunctivitis, giant papillaryconjunctivitis and contact allergies), such as asthma (particularlyallergic asthma) or other respiratory problems. Other conditions, inwhich immune suppression is desired (including, for example, organtransplantation), may also be treatable using a protein (or antagoniststhereof) of the present invention. The therapeutic effects of thepolypeptides or antagonists thereof on allergic reactions can beevaluated by in vivo animals models such as the cumulative contactenhancement test (Lastbom, et al., Toxicology 125: 59-66 (1998)), skinprick test (Hoffmann, et al., Allergy 54: 446-54 (1999)), guinea pigskin sensitization test (Vohr, et al., Arch. Toxocol. 73: 501-9), andmurine local lymph node assay (Kimber, et al., J. Toxicol. Environ.Health 53: 563-79).

Using the proteins of the invention it may also be possible to modulateimmune responses, in a number of ways. Down regulation may be in theform of inhibiting or blocking an immune response already in progress ormay involve preventing the induction of an immune response. Thefunctions of activated T cells may be inhibited by suppressing T cellresponses or by inducing specific tolerance in T cells, or both.Immunosuppression of T cell responses is generally an active,non-antigen-specific, process which requires continuous exposure of theT cells to the suppressive agent. Tolerance, which involves inducingnon-responsiveness or anergy in T cells, is distinguishable fromimmunosuppression in that it is generally antigen-specific and persistsafter exposure to the tolerizing agent has ceased. Operationally,tolerance can be demonstrated by the lack of a T cell response uponreexposure to specific antigen in the absence of the tolerizing agent.

Down regulating or preventing one or more antigen functions (includingwithout limitation B lymphocyte antigen functions (such as, for example,B7)), e.g., preventing high level lymphokine synthesis by activated Tcells, will be useful in situations of tissue, skin and organtransplantation and in graft-versus-host disease (GVHD). For example,blockage of T cell function should result in reduced tissue destructionin tissue transplantation. Typically, in tissue transplants, rejectionof the transplant is initiated through its recognition as foreign by Tcells, followed by an immune reaction that destroys the transplant. Theadministration of a therapeutic composition of the invention may preventcytokine synthesis by immune cells, such as T cells, and thus acts as animmunosuppressant. Moreover, a lack of costimulation may also besufficient to anergize the T cells, thereby inducing tolerance in asubject. Induction of long-term tolerance by B lymphocyteantigen-blocking reagents may avoid the necessity of repeatedadministration of these blocking reagents. To achieve sufficientimmunosuppression or tolerance in a subject, it may also be necessary toblock the function of a combination of B lymphocyte antigens.

The efficacy of particular therapeutic compositions in preventing organtransplant rejection or GVHD can be assessed using animal models thatare predictive of efficacy in humans. Examples of appropriate systemswhich can be used include allogeneic cardiac grafts in rats andxenogeneic pancreatic islet cell grafts in mice, both of which have beenused to examine the immunosuppressive effects of CTLA4Ig fusion proteinsin vivo as described in Lenschow, et al., Science 257:789-792 (1992) andTurka, et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). Inaddition, murine models of GVHD (see Paul ed., Fundamental Immunology,Raven Press, New York, 1989, pp. 846-847) can be used to determine theeffect of therapeutic compositions of the invention on the developmentof that disease.

Blocking antigen function may also be therapeutically useful fortreating autoimmune diseases. Many autoimmune disorders are the resultof inappropriate activation of T cells that are reactive against selftissue and which promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. Preventing the activation ofautoreactive T cells may reduce or eliminate disease symptoms.Administration of reagents which block stimulation of T cells can beused to inhibit T cell activation and prevent production ofautoantibodies or T cell-derived cytokines which may be involved in thedisease process. Additionally, blocking reagents may induceantigen-specific tolerance of autoreactive T cells which could lead tolong-term relief from the disease. The efficacy of blocking reagents inpreventing or alleviating autoimmune disorders can be determined using anumber of well-characterized animal models of human autoimmune diseases.Examples include murine experimental autoimmune encephalitis, systemiclupus erythematosus in MRL/lpr/lpr mice or NZB hybrid mice, murineautoimmune collagen arthritis, diabetes mellitus in NOD mice and BBrats, and murine experimental myasthenia gravis (see Paul ed.,Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

Upregulation of an antigen function (e.g., a B lymphocyte antigenfunction), as a means of up regulating immune responses, may also beuseful in therapy. Upregulation of immune responses may be in the formof enhancing an existing immune response or eliciting an initial immuneresponse. For example, enhancing an immune response may be useful incases of viral infection, including systemic viral diseases such asinfluenza, the common cold, and encephalitis.

Alternatively, anti-viral immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing apeptide of the present invention or together with a stimulatory form ofa soluble peptide of the present invention and reintroducing the invitro activated T cells into the patient. Another method of enhancinganti-viral immune responses would be to isolate infected cells from apatient, transfect them with a nucleic acid encoding a protein of thepresent invention as described herein such that the cells express all ora portion of the protein on their surface, and reintroduce thetransfected cells into the patient. The infected cells would now becapable of delivering a costimulatory signal to, and thereby activate, Tcells in vivo.

A polypeptide of the present invention may provide the necessarystimulation signal to T cells to induce a T cell mediated immuneresponse against the transfected tumor cells. In addition, tumor cellswhich lack MHC class I or MHC class II molecules, or which fail toreexpress sufficient mounts of MHC class I or MHC class II molecules,can be transfected with nucleic acid encoding all or a portion of (e.g.,a cytoplasmic-domain truncated portion) of an MHC class 1 alpha chainprotein and P2 microglobulin protein or an MHC class II alpha chainprotein and an MHC class II beta chain protein to thereby express MHCclass I or MHC class II proteins on the cell surface. Expression of theappropriate class I or class II MHC in conjunction with a peptide havingthe activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) inducesa T cell mediated immune response against the transfected tumor cell.Optionally, a gene encoding an antisense construct which blocksexpression of an MHC class II associated protein, such as the invariantchain, can also be cotransfected with a DNA encoding a peptide havingthe activity of a B lymphocyte antigen to promote presentation of tumorassociated antigens and induce tumor specific immunity. Thus, theinduction of a T cell mediated immune response in a human subject may besufficient to overcome tumor-specific tolerance in the subject.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for thymocyte or splenocyte cytotoxicity include,without limitation, those described in: Current Protocols in Immunology,Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience(Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19;Chapter 7, Immunologic studies in Humans); Herrmann, et al., Proc. Natl.Acad. Sci. USA 78:2488-2492 (1981); Herrmann, et al., J. Immunol.128:1968-1974 (1982); Handa, et al., J. Immunol. 135:1564-1572 (1985);Takai, et al., I. Immunol. 137:3494-3500 (1986); Takai, et al., J.Immunol. 140:508-512 (1988); Bowman, et al., J. Virology 61:1992-1998;Bertagnolli, et al., Cellular Immunology 133:327-341 (1991); Brown, etal., J. Immunol. 153:3079-3092 (1994).

Assays for T-cell-dependent immunoglobulin responses and isotypeswitching (which will identify, among others, proteins that modulateT-cell dependent antibody responses and that affect Th1/Th2 profiles)include, without limitation, those described in: Maliszewski, J.Immunol. 144:3028-3033 (1990); and Assays for B cell function: In vitroantibody production, Mond, J. J. and Brunswick, M. In Current Protocolsin Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, JohnWiley and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, amongothers, proteins that generate predominantly Th1 and CTL responses)include, without limitation, those described in: Current Protocols inImmunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.Shevach, W. Strober, Pub. Greene Publishing Associates andWiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai, etal., J. Immunol. 137:3494-3500 (1986); Takai, et al., J. Immunol.140:508-512 (1988); Bertagnolli, et al., J. Immunol. 149:3778-3783(1992).

Dendritic cell-dependent assays (which will identify, among others,proteins expressed by dendritic cells that activate naive T-cells)include, without limitation, those described in: Guery et al., J.Immunol. 134:536-544 (1995); Inaba et al., J. Exp. Med. 173:549-559(1991); Macatonia, et al., J. Immunol. 154:5071-5079 (1995); Porgador,et al., J. Exp. Med. 182:255-260 (1995); Nair, et al., J Virology67:4062-4069 (1993); Huang, et al., Science 264:961-965 (1994);Macatonia, et al., J. Exp. Med. 169:1255-1264 (1989); Bhardwaj, et al.,J. Clin. Invest. 94:797-807 (1994); and Inaba, et al., J. Exp. Med.172:631-640 (1990).

Assays for lymphocyte survival/apoptosis (which will identify, amongothers, proteins that prevent apoptosis after superantigen induction andproteins that regulate lymphocyte homeostasis) include, withoutlimitation, those described in: Darzynkiewicz et al., Cytometry13:795-808 (1992); Gorczyca, et al., Leukemia 7:659-670 (1993);Gorczyca, et al., Cancer Res. 53:1945-1951 (1993); Itoh, et al., Cell66:233-243 (1991); Zacharchuk, J. Immunol. 145:4037-4045 (1990); Zamai,et al., Cytometry 14:891-897 (1993); Gorczyca, et al., Int. J. Oncol.1:639-648 (1992).

Assays for proteins that influence early steps of T-cell commitment anddevelopment include, without limitation, those described in: Antica, etal., Blood 84:111-117 (1994); Fine, et al., Cell. Immunol. 155:111-122,(1994); Galy, et al., Blood 85:2770-2778 (1995); Toki, et al., Proc.Nat. Acad. Sci. USA 88:7548-7551 (1991).

4.17.7 Chemotactic/Chemokinetic Activity

A polypeptide of the present invention may be involved in chemotactic orchemokinetic activity for mammalian cells, including, for example,monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils,epithelial and/or endothelial cells. A polynucleotide of the inventioncan encode a polypeptide exhibiting such attributes. Chemotactic andchemokinetic receptor activation can be used to mobilize or attract adesired cell population to a desired site of action. Chemotactic orchemokinetic compositions (e.g. proteins, antibodies, binding partners,or modulators of the invention) provide particular advantages intreatment of wounds and other trauma to tissues, as well as in treatmentof localized infections. For example, attraction of lymphocytes,monocytes or neutrophils to tumors or sites of infection may result inimproved immune responses against the tumor or infecting agent.

A protein or peptide has chemotactic activity for a particular cellpopulation if it can stimulate, directly or indirectly, the directedorientation or movement of such cell population. Preferably, the proteinor peptide has the ability to directly stimulate directed movement ofcells. Whether a particular protein has chemotactic activity for apopulation of cells can be readily determined by employing such proteinor peptide in any known assay for cell chemotaxis.

Therapeutic compositions of the invention can be used in the following:

Assays for chemotactic activity (which will identify proteins thatinduce or prevent chemotaxis) consist of assays that measure the abilityof a protein to induce the migration of cells across a membrane as wellas the ability of a protein to induce the adhesion of one cellpopulation to another cell population. Suitable assays for movement andadhesion include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 6.12, Measurement of alpha and betaChemokines 6.12.1-6.12.28; Taub, et al. J. Clin. Invest. 95:1370-1376(1995); Lind, et al. APMIS 103:140-146 (1995); Muller, et al Eur. J.Immunol. 25:1744-1748; Gruber, et al. J. Immunol. 152:5860-5867 (1994);Johnston, et al. J. Immunol. 153:1762-1768 (1994).

4.17.8 Activin/Inhibin Activity

A polypeptide of the present invention may also exhibit activin- orinhibin-related activities. A polynucleotide of the invention may encodea polypeptide exhibiting such characteristics. Inhibins arecharacterized by their ability to inhibit the release of folliclestimulating hormone (FSH), while activins and are characterized by theirability to stimulate the release of follicle stimulating hormone (FSH).Thus, a polypeptide of the present invention, alone or in heterodimerswith a member of the inhibin family, may be useful as a contraceptivebased on the ability of inhibins to decrease fertility in female mammalsand decrease spermatogenesis in male mammals. Administration ofsufficient amounts of other inhibins can induce infertility in thesemammals. Alternatively, the polypeptide of the invention, as a homodimeror as a heterodimer with other protein subunits of the inhibin group,may be useful as a fertility inducing therapeutic, based upon theability of activin molecules in stimulating FSH release from cells ofthe anterior pituitary. See, for example, U.S. Pat. No. 4,798,885. Apolypeptide of the invention may also be useful for advancement of theonset of fertility in sexually immature mammals, so as to increase thelifetime reproductive performance of domestic animals such as, but notlimited to, cows, sheep and pigs.

The activity of a polypeptide of the invention may, among other means,be measured by the following methods.

Assays for activin/inhibin activity include, without limitation, thosedescribed in: Vale et al., Endocrinology 91:562-572 (1972); Ling et al.,Nature 321:779-782 (1986); Vale et al., Nature 321:776-779 (1986); Masonet al., Nature 318:659-663 (1985); Forage et al., Proc. Natl. Acad. Sci.USA 83:3091-3095 (1986).

4.17.9 Hemostatic and Thrombolytic Activity

A polypeptide of the invention may also be involved in hemostatis orthrombolysis or thrombosis. A polynucleotide of the invention can encodea polypeptide exhibiting such attributes. Compositions may be useful intreatment of various coagulation disorders (including hereditarydisorders, such as hemophilias) or to enhance coagulation and otherhemostatic events in treating wounds resulting from trauma, surgery orother causes. A composition of the invention may also be useful fordissolving or inhibiting formation of thromboses and for treatment andprevention of conditions resulting therefrom (such as, for example,infarction of cardiac and central nervous system vessels (e.g., stroke).

Therapeutic compositions of the invention can be used in the following:

Assay for hemostatic and thrombolytic activity include, withoutlimitation, those described in: Linet, et al., J. Clin. Pharmacol.26:131-140 (1986); Burdick, et al., Thrombosis Res. 45:413-419 (1987);Humphrey, et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins35:467-474 (1988).

4.17.10 Cancer Diagnosis and Therapy

Polypeptides of the invention may be involved in cancer cell generation,proliferation or metastasis. Detection of the presence or amount ofpolynucleotides or polypeptides of the invention may be useful for thediagnosis and/or prognosis of one or more types of cancer. For example,the presence or increased expression of a polynucleotide/polypeptide ofthe invention may indicate a hereditary risk of cancer, a precancerouscondition, or an ongoing malignancy. Conversely, a defect in the gene orabsence of the polypeptide may be associated with a cancer condition.Identification of single nucleotide polymorphisms associated with canceror a predisposition to cancer may also be useful for diagnosis orprognosis.

Cancer treatments promote tumor regression by inhibiting tumor cellproliferation, inhibiting angiogenesis (growth of new blood vessels thatis necessary to support tumor growth) and/or prohibiting metastasis byreducing tumor cell motility or invasiveness. Therapeutic compositionsof the invention may be effective in adult and pediatric oncologyincluding in solid phase tumors/malignancies, locally advanced tumors,human soft tissue sarcomas, metastatic cancer, including lymphaticmetastases, blood cell malignancies including multiple myeloma, acuteand chronic leukemias, and lymphomas, head and neck cancers includingmouth cancer, larynx cancer and thyroid cancer, lung cancers includingsmall cell carcinoma and non-small cell cancers, breast cancersincluding small cell carcinoma and ductal carcinoma, gastrointestinalcancers including esophageal cancer, stomach cancer, colon cancer,colorectal cancer and polyps associated with colorectal neoplasia,pancreatic cancers, liver cancer, urologic cancers including bladdercancer and prostate cancer, malignancies of the female genital tractincluding ovarian carcinoma, uterine (including endometrial) cancers,and solid tumor in the ovarian follicle, kidney cancers including renalcell carcinoma, brain cancers including intrinsic brain tumors,neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cellinvasion in the central nervous system, bone cancers including osteomas,skin cancers including malignant melanoma, tumor progression of humanskin keratinocytes, squamous cell carcinoma, basal cell carcinoma,hemangiopericytoma and Karposi's sarcoma.

Polypeptides, polynucleotides, or modulators of polypeptides of theinvention (including inhibitors and stimulators of the biologicalactivity of the polypeptide of the invention) may be administered totreat cancer. Therapeutic compositions can be administered intherapeutically effective dosages alone or in combination with adjuvantcancer therapy such as surgery, chemotherapy, radiotherapy,thermotherapy, and laser therapy, and may provide a beneficial effect,e.g. reducing tumor size, slowing rate of tumor growth, inhibitingmetastasis, or otherwise improving overall clinical condition, withoutnecessarily eradicating the cancer.

The composition can also be administered in therapeutically effectiveamounts as a portion of an anti-cancer cocktail. An anti-cancer cocktailis a mixture of the polypeptide or modulator of the invention with oneor more anti-cancer drugs in addition to a pharmaceutically acceptablecarrier for delivery. The use of anti-cancer cocktails as a cancertreatment is routine. Anti-cancer drugs that are well known in the artand can be used as a treatment in combination with the polypeptide ormodulator of the invention include: Actinomycin D, Aminoglutethimide,Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine,Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl(Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl,Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213),Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea(hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, InterferonAlpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine,Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine,Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide,Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate,Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate,Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone,Pentostatin, Semustine, Teniposide, and Vindesine sulfate.

In addition, therapeutic compositions of the invention may be used forprophylactic treatment of cancer. There are hereditary conditions and/orenvironmental situations (e.g. exposure to carcinogens) known in the artthat predispose an individual to developing cancers. Under thesecircumstances, it may be beneficial to treat these individuals withtherapeutically effective doses of the polypeptide of the invention toreduce the risk of developing cancers.

In vitro models can be used to determine the effective doses of thepolypeptide of the invention as a potential cancer treatment. These invitro models include proliferation assays of cultured tumor cells,growth of cultured tumor cells in soft agar (see Freshney, (1987)Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, NewYork, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described inGiovanella, et al., J. Natl. Can. Inst., 52: 921-30 (1974), mobility andinvasive potential of tumor cells in Boyden Chamber assays as describedin Pilkington, et al., Anticancer Res., 17: 4107-9 (1997), andangiogenesis assays such as induction of vascularization of the chickchorioallantoic membrane or induction of vascular endothelial cellmigration as described in Ribatta, et al., Intl. J. Dev. Biol., 40:1189-97 (1999) and Li, et al., Clin. Exp. Metastasis, 17:423-9 (1999),respectively. Suitable tumor cells lines are available, e.g. fromAmerican Type Tissue Culture Collection catalogs.

4.17.11 Receptor/Ligand Activity

A polypeptide of the present invention may also demonstrate activity asreceptor, receptor ligand or inhibitor or agonist of receptor/ligandinteractions. A polynucleotide of the invention can encode a polypeptideexhibiting such characteristics. Examples of such receptors and ligandsinclude, without limitation, cytokine receptors and their ligands,receptor kinases and their ligands, receptor phosphatases and theirligands, receptors involved in cell-cell interactions and their ligands(including without limitation, cellular adhesion molecules (such asselectins, integrins and their ligands) and receptor/ligand pairsinvolved in antigen presentation, antigen recognition and development ofcellular and humoral immune responses. Receptors and ligands are alsouseful for screening of potential peptide or small molecule inhibitorsof the relevant receptor/ligand interaction. A protein of the presentinvention (including, without limitation, fragments of receptors andligands) may themselves be useful as inhibitors of receptor/ligandinteractions.

The activity of a polypeptide of the invention may, among other means,be measured by the following methods:

Suitable assays for receptor-ligand activity include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28,Measurement of Cellular Adhesion under static conditions7.28.1-7.28.22), Takai, et al., Proc. Natl. Acad. Sci. USA 84:6864-6868(1987); Bierer, et al., J. Exp. Med. 168:1145-1156 (1988); Rosenstein,et al., J. Exp. Med. 169:149-160 (1989); Stoltenborg, et al., J.Immunol. Methods 175:59-68 (1994); Stitt, et al., Cell 80:661-670(1995).

By way of example, the polypeptides of the invention may be used as areceptor for a ligand(s) thereby transmitting the biological activity ofthat ligand(s). Ligands may be identified through binding assays,affinity chromatography, dihybrid screening assays, BIAcore assays, geloverlay assays, or other methods known in the art.

Studies characterizing drugs or proteins as agonist or antagonist orpartial agonists or a partial antagonist require the use of otherproteins as competing ligands. The polypeptides of the present inventionor ligand(s) thereof may be labeled by being coupled to radioisotopes,calorimetric molecules or a toxin molecules by conventional methods.(“Guide to Protein Purification” Murray P. Deutscher (ed) Methods inEnzymology Vol. 182 (1990) Academic Press, Inc. San Diego). Examples ofradioisotopes include, but are not limited to, tritium and carbon-14.Examples of calorimetric molecules include, but are not limited to,fluorescent molecules such as fluorescamine, or rhodamine or othercolorimetric molecules. Examples of toxins include, but are not limited,to ricin.

4.17.12 Drug Screening

This invention is particularly useful for screening chemical compoundsby using the novel polypeptides or binding fragments thereof in any of avariety of drug screening techniques. The polypeptides or fragmentsemployed in such a test may either be free in solution, affixed to asolid support, borne on a cell surface or located intracellularly. Onemethod of drug screening utilizes eukaryotic or prokaryotic host cellswhich are stably transformed with recombinant nucleic acids expressingthe polypeptide or a fragment thereof. Drugs are screened against suchtransformed cells in competitive binding assays. Such cells, either inviable or fixed form, can be used for standard binding assays. One maymeasure, for example, the formation of complexes between polypeptides ofthe invention or fragments and the agent being tested or examine thediminution in complex formation between the novel polypeptides and anappropriate cell line, which are well known in the art.

Sources for test compounds that may be screened for ability to bind toor modulate (i.e., increase or decrease) the activity of polypeptides ofthe invention include (1) inorganic and organic chemical libraries, (2)natural product libraries, and (3) combinatorial libraries comprised ofeither random or mimetic peptides, oligonucleotides or organicmolecules.

Chemical libraries may be readily synthesized or purchased from a numberof commercial sources, and may include structural analogs of knowncompounds or compounds that are identified as “hits” or “leads” vianatural product screening.

The sources of natural product libraries are microorganisms (includingbacteria and fingi), animals, plants or other vegetation, or marineorganisms, and libraries of mixtures for screening may be created by:(1) fermentation and extraction of broths from soil, plant or marinemicroorganisms or (2) extraction of the organisms themselves. Naturalproduct libraries include polyketides, non-ribosomal peptides, and(non-naturally occurring) variants thereof. For a review, see Science282:63-68 (1998).

Combinatorial libraries are composed of large numbers of peptides,oligonucleotides or organic compounds and can be readily prepared bytraditional automated synthesis methods, PCR, cloning or proprietarysynthetic methods. Of particular interest are peptide andoligonucleotide combinatorial libraries. Still other libraries ofinterest include peptide, protein, peptidomimetic, multiparallelsynthetic collection, recombinatorial, and polypeptide libraries. For areview of combinatorial chemistry and libraries created therefrom, seeMyers, Curr. Opin. Biotechnol. 8:701-707 (1997). For reviews andexamples of peptidomimetic libraries, see Al-Obeidi et al., Mol.Biotechnol, 9:205-23 (1998); Hruby, et al., Curr Opin Chem Biol,1:114-19 (1997); Dorner, et al., Bioorg Med Chem, 4:709-15 (1996)(alkylated dipeptides).

Identification of modulators through use of the various librariesdescribed herein permits modification of the candidate “hit” (or “lead”)to optimize the capacity of the “hit” to bind a polypeptide of theinvention. The molecules identified in the binding assay are then testedfor antagonist or agonist activity in in vivo tissue culture or animalmodels that are well known in the art. In brief, the molecules aretitrated into a plurality of cell cultures or animals and then testedfor either cell/animal death or prolonged survival of the animal/cells.

The binding molecules thus identified may be complexed with toxins,e.g., ricin or cholera, or with other compounds that are toxic to cellssuch as radioisotopes. The toxin-binding molecule complex is thentargeted to a tumor or other cell by the specificity of the bindingmolecule for a polypeptide of the invention. Alternatively, the bindingmolecules may be complexed with imaging agents for targeting and imagingpurposes.

4.17.13 Assay for Receptor Activity

The invention also provides methods to detect specific binding of apolypeptide e.g. a ligand or a receptor. The invention also providesmethods to detect specific binding of a polypeptide of the invention toa binding partner polypeptide, and in particular a ligand polypeptide.Ligands useful in binding assays of this type include, for exampleNogo-A, Nogo-B, Nogo-C, and Nogo-66 or related protein for NgRHy, andother binding partner/receptors for other polypeptides of the inventionidentified using assays well known and routinely practiced in the art.

In one embodiment, receptor activity of the polypeptides of theinvention is determined using a method that involves (1) forming amixture comprising a polypeptide of the invention, and/or its agonistsand antagonists (or agonist or antagonist drug candidates) and/orantibodies specific for the polypeptides of the invention; (2)incubating the mixture under conditions whereby, but for the presence ofsaid polypeptide of the invention and/or agonists and antagonists (oragonist or antagonist drug candidates) and/or antibodies specific forthe polypeptides of the invention, the ligand binds to the receptor; and(3) detecting the presence or absence of specific binding of thepolypeptide of the invention to its ligand.

The art provides numerous assays particularly useful for identifyingpreviously unknown binding partners for receptor polypeptides of theinvention. For example, expression cloning using mammalian or bacterialcells, or dihybrid screening assays can be used to identifypolynucleotides encoding binding partners. As another example, affinitychromatography with the appropriate immobilized polypeptide of theinvention can be used to isolate polypeptides that recognize and bindpolypeptides of the invention. There are a number of different librariesused for the identification of compounds, and in particular smallmolecules, that modulate (i.e., increase or decrease) biologicalactivity of a polypeptide of the invention. Ligands for receptorpolypeptides of the invention can also be identified by adding exogenousligands, or cocktails of ligands to two cells populations that aregenetically identical except for the expression of the receptor of theinvention: one cell population expresses the receptor of the inventionwhereas the other does not. The response of the two cell populations tothe addition of ligands(s) is then compared. Alternatively, anexpression library can be co-expressed with the polypeptide of theinvention in cells and assayed for an autocrine response to identifypotential ligand(s). As still another example, BIAcore assays, geloverlay assays, or other methods known in the art can be used toidentify binding partner polypeptides, including, (1) organic andinorganic chemical libraries, (2) natural product libraries, and (3)combinatorial libraries comprised of random peptides, oligonucleotidesor organic molecules.

The role of downstream intracellular signaling molecules in thesignaling cascade of the polypeptide of the invention can be determined.For example, a chimeric protein in which the cytoplasmic domain of thepolypeptide of the invention is fused to the extracellular portion of aprotein, whose ligand has been identified, is produced in a host cell.The cell is then incubated with the ligand specific for theextracellular portion of the chimeric protein, thereby activating thechimeric receptor. Known downstream proteins involved in intracellularsignaling can then be assayed for expected modifications i.e.phosphorylation. Other methods known to those in the art can also beused to identify signaling molecules involved in receptor activity.

4.17.14 Leukemia

Leukemia and related disorders may be treated or prevented byadministration of a therapeutic that promotes or inhibits function ofthe polynucleotides and/or polypeptides of the invention. Such leukemiasand related disorders include but are not limited to acute leukemia,acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic,promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronicleukemia, chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia (for a review of such disorders, see Fishman, etal., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).

4.17.15 Nervous System Disorders

Nervous system disorders, involving cell types which can be tested forefficacy of intervention with compounds that modulate the activity ofthe polynucleotides and/or polypeptides of the invention, and which canbe treated upon thus observing an indication of therapeutic utility,include but are not limited to nervous system injuries, and diseases ordisorders which result in either a disconnection of axons, a diminutionor degeneration of neurons, or demyelination. Nervous system lesionswhich may be treated in a patient (including human and non-humanmammalian patients) according to the invention include but are notlimited to the following lesions of either the central (including spinalcord, brain) or peripheral nervous systems:

-   -   (i) traumatic lesions, including lesions caused by physical        injury or associated with surgery, for example, lesions which        sever a portion of the nervous system, or compression injuries;    -   (ii) ischemic lesions, in which a lack of oxygen in a portion of        the nervous system results in neuronal injury or death,        including cerebral infarction or ischemia, or spinal cord        infarction or ischemia;    -   (iii) infectious lesions, in which a portion of the nervous        system is destroyed or injured as a result of infection, for        example, by an abscess or associated with infection by human        immunodeficiency virus, herpes zoster, or herpes simplex virus        or with Lyme disease, tuberculosis, syphilis;    -   (iv) degenerative lesions, in which a portion of the nervous        system is destroyed or injured as a result of a degenerative        process including but not limited to degeneration associated        with Parkinson's disease, Alzheimer's disease, Huntington's        chorea, or amyotrophic lateral sclerosis;    -   (v) lesions associated with nutritional diseases or disorders,        in which a portion of the nervous system is destroyed or injured        by a nutritional disorder or disorder of metabolism including        but not limited to, vitamin B12 deficiency, folic acid        deficiency, Wernicke disease, tobacco-alcohol amblyopia,        Marchiafava-Bignami disease (primary degeneration of the corpus        callosum), and alcoholic cerebellar degeneration;    -   (vi) neurological lesions associated with systemic diseases        including but not limited to diabetes (diabetic neuropathy,        Bell's palsy), systemic lupus erythematosus, carcinoma, or        sarcoidosis;    -   (vii) lesions caused by toxic substances including alcohol,        lead, or particular neurotoxins; and    -   (viii) demyelinated lesions in which a portion of the nervous        system is destroyed or injured by a demyelinating disease        including but not limited to multiple sclerosis, monophasic        demyelination, encephalomyelitis, panencephalaitis,        Marchiafava-Bignami disease, Spongy degeneration, Alexander's        disease, Canavan's disease, metachromatic leukodystrophy,        Krabbe's disease, human immunodeficiency virus-associated        myelopathy, transverse myelopathy or various etiologies,        progressive multifocal leukoencephalopathy, Guillain-Barre        Syndrome, and central pontine myelinolysis.

Therapeutics which are useful according to the invention for treatmentof a nervous system disorder may be selected by testing for biologicalactivity in promoting the survival or differentiation of neurons. Forexample, and not by way of limitation, therapeutics which elicit any ofthe following effects may be useful according to the invention:

-   -   (i) increased survival time of neurons in culture;    -   (ii) increased sprouting of neurons in culture or in vivo;    -   (iii) increased production of a neuron-associated molecule in        culture or in vivo, e.g., choline acetyltransferase or        acetylcholinesterase with respect to motor neurons; or    -   (iv) decreased symptoms of neuron dysfunction in vivo.

Such effects may be measured by any method known in the art. Inpreferred, nonlimiting embodiments, increased survival of neurons may bemeasured by the method set forth in Arakawa et al. (J. Neurosci.10:3507-3515 (1990)); increased sprouting of neurons may be detected bymethods set forth in Pestronk, et al. (Exp. Neurol. 70:65-82 (1980)) orBrown, et al. (Ann. Rev. Neurosci. 4:17-42 (1981)); increased productionof neuron-associated molecules may be measured by bioassay, enzymaticassay, antibody binding, Northern blot assay, etc., depending on themolecule to be measured; and motor neuron dysfunction may be measured byassessing the physical manifestation of motor neuron disorder, e.g.,weakness, motor neuron conduction velocity, or functional disability.

In specific embodiments, motor neuron disorders that may be treatedaccording to the invention include but are not limited to disorders suchas infarction, infection, exposure to toxin, trauma, surgical damage,degenerative disease or malignancy that may affect motor neurons as wellas other components of the nervous system, as well as disorders thatselectively affect neurons such as amyotrophic lateral sclerosis, andincluding but not limited to progressive spinal muscular atrophy,progressive bulbar palsy, primary lateral sclerosis, infantile andjuvenile muscular atrophy, progressive bulbar paralysis of childhood(Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, andHereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

4.17.16 Other Activities

A polypeptide of the invention may also exhibit one or more of thefollowing additional activities or effects: inhibiting the growth,infection or function of, or killing, infectious agents, including,without limitation, bacteria, viruses, fungi and other parasites;effecting (suppressing or enhancing) bodily characteristics, including,without limitation, height, weight, hair color, eye color, skin, fat tolean ratio or other tissue pigmentation, or organ or body part size orshape (such as, for example, breast augmentation or diminution, changein bone form or shape); effecting biorhythms or circadian cycles orrhythms; effecting the fertility of male or female subjects; effectingthe metabolism, catabolism, anabolism, processing, utilization, storageor elimination of dietary fat, lipid, protein, carbohydrate, vitamins,minerals, co-factors or other nutritional factors or component(s);effecting behavioral characteristics, including, without limitation,appetite, libido, stress, cognition (including cognitive disorders),depression (including depressive disorders) and violent behaviors;providing analgesic effects or other pain reducing effects; promotingdifferentiation and growth of embryonic stem cells in lineages otherthan hematopoietic lineages; hormonal or endocrine activity; in the caseof enzymes, correcting deficiencies of the enzyme and treatingdeficiency-related diseases; treatment of hyperproliferative disorders(such as, for example, psoriasis); immunoglobulin-like activity (suchas, for example, the ability to bind antigens or complement); and theability to act as an antigen in a vaccine composition to raise an immuneresponse against such protein or another material or entity which iscross-reactive with such protein.

4.17.17 Identification of Polymorphisms

The demonstration of polymorphisms makes possible the identification ofsuch polymorphisms in human subjects and the pharmacogenetic use of thisinformation for diagnosis and treatment. Such polymorphisms may beassociated with, e.g., differential predisposition or susceptibility tovarious disease states (such as disorders involving inflammation orimmune response) or a differential response to drug administration, andthis genetic information can be used to tailor preventive or therapeutictreatment appropriately. For example, the existence of a polymorphismassociated with a predisposition to inflammation or autoimmune diseasemakes possible the diagnosis of this condition in humans by identifyingthe presence of the polymorphism.

Polymorphisms can be identified in a variety of ways known in the artwhich all generally involve obtaining a sample from a patient, analyzingDNA from the sample, optionally involving isolation or amplification ofthe DNA, and identifying the presence of the polymorphism in the DNA.For example, PCR may be used to amplify an appropriate fragment ofgenomic DNA which may then be sequenced. Alternatively, the DNA may besubjected to allele-specific oligonucleotide hybridization (in whichappropriate oligonucleotides are hybridized to the DNA under conditionspermitting detection of a single base mismatch) or to a singlenucleotide extension assay (in which an oligonucleotide that hybridizesimmediately adjacent to the position of the polymorphism is extendedwith one or more labeled nucleotides). In addition, traditionalrestriction fragment length polymorphism analysis (using restrictionenzymes that provide differential digestion of the genomic DNA dependingon the presence or absence of the polymorphism) may be performed. Arrayswith nucleotide sequences of the present invention can be used to detectpolymorphisms. The array can comprise modified nucleotide sequences ofthe present invention in order to detect the nucleotide sequences of thepresent invention. In the alternative, any one of the nucleotidesequences of the present invention can be placed on the array to detectchanges from those sequences.

Alternatively a polymorphism resulting in a change in the amino acidsequence could also be detected by detecting a corresponding change inamino acid sequence of the protein, e.g., by an antibody specific to thevariant sequence.

4.17.18 Arthritis and Inflammation

The immunosuppressive effects of the compositions of the inventionagainst rheumatoid arthritis are determined in an experimental animalmodel system. The experimental model system is adjuvant inducedarthritis in rats, and the protocol is described by J. Holoshitz, etal., Science, 219:56 (1983), or by B. Waksman, et al., Int. Arch.Allergy Appl. Immunol., 23:129 (1963). Induction of the disease can becaused by a single injection, generally intradermally, of a suspensionof killed Mycobacterium tuberculosis in complete Freund's adjuvant(CFA). The route of injection can vary, but rats may be injected at thebase of the tail with an adjuvant mixture. The polypeptide isadministered in phosphate buffered solution (PBS) at a dose of about 1-5mg/kg. The control consists of administering PBS only.

The procedure for testing the effects of the test compound would consistof intradermally injecting killed Mycobacterium tuberculosis in CFAfollowed by immediately administering the test compound and subsequenttreatment every other day until day 24. At 14, 15, 18, 20, 22, and 24days after injection of Mycobacterium CFA, an overall arthritis scoremay be obtained as described by J. Holoskitz above. An analysis of thedata would reveal that the test compound would have a dramatic affect onthe swelling of the joints as measured by a decrease of the arthritisscore.

Compositions of the present invention may also exhibit otheranti-inflammatory activity. The anti-inflammatory activity may beachieved by providing a stimulus to cells involved in the inflammatoryresponse, by inhibiting or promoting cell-cell interactions (such as,for example, cell adhesion), by inhibiting or promoting chemotaxis ofcells involved in the inflammatory process, inhibiting or promoting cellextravasation, or by stimulating or suppressing production of otherfactors which more directly inhibit or promote an inflammatory response.Compositions with such activities can be used to treat inflammatoryconditions including chronic or acute conditions), including withoutlimitation intimation associated with infection (such as septic shock,sepsis or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine-induced lung injury, inflammatory bowel disease, Crohn'sdisease or resulting from over production of cytokines such as TNF orIL-1. Compositions of the invention may also be useful to treatanaphylaxis and hypersensitivity to an antigenic substance or material.Compositions of this invention may be utilized to prevent or treatconditions such as, but not limited to, sepsis, acute pancreatitis,endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronicinflammatory arthritis, pancreatic cell damage from diabetes mellitustype 1, graft versus host disease, inflammatory bowel disease,inflamation associated with pulmonary disease, other autoimmune diseaseor inflammatory disease, or in the prevention of premature laborsecondary to intrauterine infections.

4.17.19 Nutritional Uses

Polynucleotides and polypeptides of the present invention can also beused as nutritional sources or supplements. Such uses include withoutlimitation use as a protein or amino acid supplement, use as a carbonsource, use as a nitrogen source and use as a source of carbohydrate. Insuch cases the polypeptide or polynucleotide of the invention can beadded to the feed of a particular organism or can be administered as aseparate solid or liquid preparation, such as in the form of powder,pills, solutions, suspensions or capsules. In the case ofmicroorganisms, the polypeptide or polynucleotide of the invention canbe added to the medium in or on which the microorganism is cultured.Additionally, the polypeptides of the invention can be used as markers,and as a food supplement. Protein food supplements are well known andthe formulation of suitable food supplements including polypeptides ofthe invention is within the level of skill in the food preparation art.

4.17.20 Metabolic Disorders

A polynucleotide and polypeptide of the invention may also be involvedin the prevention, diagnosis and management of metabolic disordersinvolving carbohydrates, lipids, amino acids, vitamins etc., includingbut not limited to diabetes mellitus, obesity, aspartylglusomarinuria,carbohydrate deficient glycoprotein syndrome (CDGS), cystinosis,diabetes insipidus, Fabry, fatty acid metabolism disorders,galactosemia, Gaucher, glucose-6-phosphate dehydrogenase (G6PD),glutaric aciduria, Hurler, Hurler-Scheie, Hunter, hypophosphatemia,1-cell, Krabbe, lactic acidosis, long chain 3 hydroxyacyl CoAdehydrogenase deficiency (LCHAD), lysosomal storage diseases,mannosidosis, maple syrup urine, Maroteaux-Lamy, metachromaticleukodystrophy, mitochondrial Morquio, mucopolysaccharidosis,neuro-metabolic, Niemann-Pick, organic acidemias, purine,phenylketonuria (PKU), Pompe, porphyria, pseudo-Hurler, pyruvatedehydrogenase deficiency, Sandhoff, Sanfilippo, Scheie, Sly, Tay-Sachs,trimethylaminuria (Fish-Malodor syndrome), urea cycle conditions,vitamin D deficiency rickets and related complications involvingdifferent organs including but not limited to liver, heart, kidney, eye,brain, muscle development etc. Hereditary and/or environmental factorsknown in the art can predispose an individual to developing metabolicdisorders and conditions resulting therefrom. Under these circumstances,it maybe beneficial to treat these individual with therapeuticallyeffective doses of the polypeptide of the invention to reduce the riskof developing the disorder. Examples of such disorders include diabetesmellitus, obesity and cardiovascular disease. Further, polynucleotidesequences encoding the invention may be used in Southern or Northernanalysis, dot blot, or other membrane-based technologies; in PCRtechnologies; or in dip stick, pin, ELISA or chip assays utilizingfluids or tissues from patient biopsies to detect altered expression ofthe polynucleotides of the invention. Such qualitative or quantitativemethods are well known in the art.

4.7.21 Cardiovascular Disease and Therapy

Polypeptides and polynucleotides of the invention may also be involvedin the prevention, diagnosis and management of cardiovascular disorderssuch as coronary artery disease, atherosclerosis and hyper- andhypolipoproteinemia, hypertension, angina pectoris, myocardialinfarction, congestive heart failure, cardiac arrythmias includingparoxysmal arrythmias, restenosis after angioplasty, aortic aneurysm andrelated complications involving various organs including but not limitedto kidney, eye, brain, heart etc. Polypeptides of the invention may alsohave direct and indirect effects on myocardial contractility, electricalactivity of the heart, atrial fibrillation, atrial fluter, anomalousatrio-ventricular pathways, sino-atrial dysfunction, vascularinsufficiency and arterial embolism. Hereditary and/or environmentalfactors known in the art can predispose an individual to developingmetabolic disorders and conditions resulting therefrom. Under thesecircumstances, it maybe beneficial to treat these individual withtherapeutically effective doses of the polypeptide of the invention toreduce the risk of developing the disorder. Examples of such disordersinclude but are not limited to coronary artery disease, atherosclerosis,hyper- and hypolipoproteinemia, hypertension, angina pectoris,myocardial infarction, cardiac arrythmias including paroxysmalarrythmias, diabetes mellitus, inflammatory glomerulonephritis, ischemicrenal failure, extracellular matrix accumulation, fibrosis,hypertension, coronary vasoconstriction, ischemic heart disease, andlesions occurring in brain disorders such as stroke, trauma, infarcts,aneurysms.

The polynucleotide sequences encoding the invention may be used inSouthern or Northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; or in dip stick, pin, ELISA or chipassays utilizing fluids or tissues from patient biopsies to detectaltered expression of the polynucleotides of the invention. Suchqualitative or quantitative methods are well known in the art.

4.18 Therapeutic Methods

The compositions (including polypeptide fragments, analogs, variants andantibodies or other binding partners or modulators including antisensepolynucleotides) of the invention have numerous applications in avariety of therapeutic methods. Examples of therapeutic applicationsinclude, but are not limited to, those exemplified herein.

4.18.1 Example

One embodiment of the invention is the administration of an effectiveamount of the polypeptides of the invention or other composition of theinvention to individuals affected by a disease or disorder that can bemodulated by regulating the peptides of the invention. While the mode ofadministration is not particularly important, parenteral administrationis preferred. An exemplary mode of administration is to deliver anintravenous bolus. The dosage of polypeptides of the invention or othercomposition of the invention will normally be determined by theprescribing physician. It is to be expected that the dosage will varyaccording to the age, weight, condition and response of the individualpatient. Typically, the amount of polypeptide administered per dose willbe in the range of about 0.01 μg/kg to 100 mg/kg of body weight, withthe preferred dose being about 0.1 μg/kg to 10 mg/kg of patient bodyweight. For parenteral administration, polypeptides of the inventionwill be formulated in an injectable form combined with apharmaceutically acceptable parenteral vehicle. Such vehicles are wellknown in the art and examples include water, saline, Ringer's solution,dextrose solution, and solutions consisting of small amounts of thehuman serum albumin. The vehicle may contain minor amounts of additivesthat maintain the isotonicity and stability of the polypeptide or otheractive ingredient. The preparation of such solutions is within the skillof the art.

4.19 Pharmaceutical Formulations and Routes of Administration

A protein or other composition of the present invention (from whateversource derived, including without limitation from recombinant andnon-recombinant sources and including antibodies and other bindingpartners of the polypeptides of the invention) may be administered to apatient in need, by itself, or in pharmaceutical compositions where itis mixed with suitable carriers or excipient(s) at doses to treat orameliorate a variety of disorders. Such a composition may optionallycontain (in addition to protein or other active ingredient and acarrier) diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials well known in the art. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The characteristics of the carrier will depend on the route ofadministration. The pharmaceutical composition of the invention may alsocontain cytokines, lymphokines, or other hematopoietic factors such asM-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2,G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. Infurther compositions, proteins of the invention may be combined withother agents beneficial to the treatment of the disease or disorder inquestion. These agents include various growth factors such as epidermalgrowth factor (EGF), platelet-derived growth factor (PDGF), transforminggrowth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), aswell as cytokines described herein.

The pharmaceutical composition may further contain other agents whicheither enhance the activity of the protein or other active ingredient orcomplement its activity or use in treatment. Such additional factorsand/or agents may be included in the pharmaceutical composition toproduce a synergistic effect with protein or other active ingredient ofthe invention, or to minimize side effects. Conversely, protein or otheractive ingredient of the present invention may be included informulations of the particular clotting factor, cytokine, lymphokine,other hematopoietic factor, thrombolytic or anti-thrombotic factor, oranti-inflammatory agent to minimize side effects of the clotting factor,cytokine, lymphokine, other hematopoietic factor, thrombolytic oranti-thrombotic factor, or antiinflammatory agent (such as IL-1Ra, IL-1Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents). Aprotein of the present invention may be active in multimers (e.g.,heterodimers or homodimers) or complexes with itself or other proteins.As a result, pharmaceutical compositions of the invention may comprise aprotein of the invention in such multimeric or complexed form.

As an alternative to being included in a pharmaceutical composition ofthe invention including a first protein, a second protein or atherapeutic agent may be concurrently administered with the firstprotein (e.g., at the same time, or at differing times provided thattherapeutic concentrations of the combination of agents is achieved atthe treatment site). Techniques for formulation and administration ofthe compounds of the instant application may be found in “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latestedition. A therapeutically effective dose further refers to that amountof the compound sufficient to result in amelioration of symptoms, e.g.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, a therapeutically effective dose refersto that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of protein or other active ingredientof the present invention is administered to a mammal having a conditionto be treated. Protein or other active ingredient of the presentinvention may be administered in accordance with the method of theinvention either alone or in combination with other therapies such astreatments employing cytokines, lymphokines or other hematopoieticfactors. When co-administered with one or more cytokines, lymphokines orother hematopoietic factors, protein or other active ingredient of thepresent invention may be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or sequentially. If administeredsequentially, the attending physician will decide on the appropriatesequence of administering protein or other active ingredient of thepresent invention in combination with cytokine(s), lymphokine(s), otherhematopoietic factor(s), thrombolytic or anti-thrombotic factors.

4.19.1 Routes of Administration

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. Administrationof protein or other active ingredient of the present invention used inthe pharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such asoral ingestion, inhalation, topical application or cutaneous,subcutaneous, intraperitoneal, parenteral or intravenous injection.Intravenous administration to the patient is preferred.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto arthritic joints or in fibrotic tissue, often in a depot orsustained release formulation. In order to prevent the scarring processfrequently occurring as complication of glaucoma surgery, the compoundsmay be administered topically, for example, as eye drops. Furthermore,one may administer the drug in a targeted drug delivery system, forexample, in a liposome coated with a specific antibody, targeting, forexample, arthritic or fibrotic tissue. The liposomes will be targeted toand taken up selectively by the afflicted tissue.

The polypeptides of the invention are administered by any route thatdelivers an effective dosage to the desired site of action. Thedetermination of a suitable route of administration and an effectivedosage for a particular indication is within the level of skill in theart. Preferably for wound treatment, one administers the therapeuticcompound directly to the site. Suitable dosage ranges for thepolypeptides of the invention can be extrapolated from these dosages orfrom similar studies in appropriate animal models. Dosages can then beadjusted as necessary by the clinician to provide maximal therapeuticbenefit.

4.19.2 Compositions/Formulations

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in a conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. These pharmaceuticalcompositions may be manufactured in a manner that is itself known, e.g.,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Proper formulation is dependent upon the route ofadministration chosen. When a therapeutically effective amount ofprotein or other active ingredient of the present invention isadministered orally, protein or other active ingredient of the presentinvention will be in the form of a tablet, capsule, powder, solution orelixir. When administered in tablet form, the pharmaceutical compositionof the invention may additionally contain a solid carrier such as agelatin or an adjuvant. The tablet, capsule, and powder contain fromabout 5 to 95% protein or other active ingredient of the presentinvention, and preferably from about 25 to 90% protein or other activeingredient of the present invention. When administered in liquid form, aliquid carrier such as water, petroleum, oils of animal or plant originsuch as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of protein or other active ingredient of the present invention,and preferably from about 1 to 50% protein or other active ingredient ofthe present invention.

When a therapeutically effective amount of protein or other activeingredient of the present invention is administered by intravenous,cutaneous or subcutaneous injection, protein or other active ingredientof the present invention will be in the form of a pyrogen-free,parenterally acceptable aqueous solution. The preparation of suchparenterally acceptable protein or other active ingredient solutions,having due regard to pH, isotonicity, stability, and the like, is withinthe skill in the art. A preferred pharmaceutical composition forintravenous, cutaneous, or subcutaneous injection should contain, inaddition to protein or other active ingredient of the present invention,an isotonic vehicle such as Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection,Lactated Ringer's Injection, or other vehicle as known in the art. Thepharmaceutical composition of the present invention may also containstabilizers, preservatives, buffers, antioxidants, or other additivesknown to those of skill in the art. For injection, the agents of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. The compounds maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. In additionto the formulations described previously, the compounds may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a cosolvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. The co-solventsystem may be the VPD co-solvent system. VPD is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5%dextrose in water solution. This co-solvent system dissolves hydrophobiccompounds well, and itself produces low toxicity upon systemicadministration. Naturally, the proportions of a co-solvent system may bevaried considerably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose. Alternatively, otherdelivery systems for hydrophobic pharmaceutical compounds may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Certain organic solventssuch as dimethylsulfoxide also may be employed, although usually at thecost of greater toxicity. Additionally, the compounds may be deliveredusing a sustained-release system, such as semipermeable matrices ofsolid hydrophobic polymers containing the therapeutic agent. Varioustypes of sustained-release materials have been established and are wellknown by those skilled in the art. Sustained-release capsules may,depending on their chemical nature, release the compounds for a fewweeks up to over 100 days. Depending on the chemical nature and thebiological stability of the therapeutic reagent, additional strategiesfor protein or other active ingredient stabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols. Many of the active ingredients of theinvention may be provided as salts with pharmaceutically compatiblecounter ions. Such pharmaceutically acceptable base addition salts arethose salts which retain the biological effectiveness and properties ofthe free acids and which are obtained by reaction with inorganic ororganic bases such as sodium hydroxide, magnesium hydroxide, ammonia,trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodiumacetate, potassium benzoate, triethanol amine and the like.

The pharmaceutical composition of the invention may be in the form of acomplex of the protein(s) or other active ingredient of presentinvention along with protein or peptide antigens. The protein and/orpeptide antigen will deliver a stimulatory signal to both B and Tlymphocytes. B lymphocytes will respond to antigen through their surfaceimmunoglobulin receptor. T lymphocytes will respond to antigen throughthe T cell receptor (TCR) following presentation of the antigen by MHCproteins. MHC and structurally related proteins including those encodedby class I and class II MHC genes on host cells will serve to presentthe peptide antigen(s) to T lymphocytes. The antigen components couldalso be supplied as purified MHC-peptide complexes alone or withco-stimulatory molecules that can directly signal T cells. Alternativelyantibodies able to bind surface immunoglobulin and other molecules on Bcells as well as antibodies able to bind the TCR and other molecules onT cells can be combined with the pharmaceutical composition of theinvention.

The pharmaceutical composition of the invention may be in the form of aliposome in which protein of the present invention is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithins,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728;4,837,028; and 4,737,323, all of which are incorporated herein byreference.

The amount of protein or other active ingredient of the presentinvention in the pharmaceutical composition of the present inventionwill depend upon the nature and severity of the condition being treated,and on the nature of prior treatments which the patient has undergone.Ultimately, the attending physician will decide the amount of protein orother active ingredient of the present invention with which to treateach individual patient. Initially, the attending physician willadminister low doses of protein or other active ingredient of thepresent invention and observe the patient's response. Larger doses ofprotein or other active ingredient of the present invention may beadministered until the optimal therapeutic effect is obtained for thepatient, and at that point the dosage is not increased further. It iscontemplated that the various pharmaceutical compositions used topractice the method of the present invention should contain about 0.01μg to about 100 mg (preferably about 0.1 μg to about 10 mg, morepreferably about 0.1 μg to about 1 mg) of protein or other activeingredient of the present invention per kg body weight. For compositionsof the present invention which are useful for bone, cartilage, tendon orligament regeneration, the therapeutic method includes administering thecomposition topically, systematically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Further, the composition may desirably be encapsulated or injectedin a viscous form for delivery to the site of bone, cartilage or tissuedamage. Topical administration may be suitable for wound healing andtissue repair. Therapeutically useful agents other than a protein orother active ingredient of the invention which may also optionally beincluded in the composition as described above, may alternatively oradditionally, be administered simultaneously or sequentially with thecomposition in the methods of the invention. Preferably for bone and/orcartilage formation, the composition would include a matrix capable ofdelivering the protein-containing or other active ingredient-containingcomposition to the site of bone and/or cartilage damage, providing astructure for the developing bone and cartilage and optimally capable ofbeing reabsorbed into the body. Such matrices may be formed of materialspresently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalcium phosphate. The bioceramics may be altered in composition,such as in calcium-aluminate-phosphate and processing to alter poresize, particle size, particle shape, and biodegradability. Presentlypreferred is a 50:50 (mole weight) copolymer of lactic acid and glycolicacid in the form of porous particles having diameters ranging from 150to 800 microns. In some applications, it will be useful to utilize asequestering agent, such as carboxymethyl cellulose or autologous bloodclot, to prevent the protein compositions from disassociating from thematrix.

A preferred family of sequestering agents is cellulosic materials suchas alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorption of the protein from the polymer matrixand to provide appropriate handling of the composition, yet not so muchthat the progenitor cells are prevented from infiltrating the matrix,thereby providing the protein the opportunity to assist the osteogenicactivity of the progenitor cells. In further compositions, proteins orother active ingredient of the invention may be combined with otheragents beneficial to the treatment of the bone and/or cartilage defect,wound, or tissue in question. These agents include various growthfactors such as epidermal growth factor (EGF), platelet derived growthfactor (PDGF), transforming growth factors (TGF-α and TGF-β), andinsulin-like growth factor (IGF).

The therapeutic compositions are also presently valuable for veterinaryapplications. Particularly domestic animals and thoroughbred horses, inaddition to humans, are desired patients for such treatment withproteins or other active ingredient of the present invention. The dosageregimen of a protein-containing pharmaceutical composition to be used intissue regeneration will be determined by the attending physicianconsidering various factors which modify the action of the proteins,e.g., amount of tissue weight desired to be formed, the site of damage,the condition of the damaged tissue, the size of a wound, type ofdamaged tissue (e.g., bone), the patient's age, sex, and diet, theseverity of any infection, time of administration and other clinicalfactors. The dosage may vary with the type of matrix used in thereconstitution and with inclusion of other proteins in thepharmaceutical composition. For example, the addition of other knowngrowth factors, such as IGF I (insulin like growth factor I), to thefinal composition, may also effect the dosage. Progress can be monitoredby periodic assessment of tissue/bone growth and/or repair, for example,X-rays, histomorphometric determinations and tetracycline labeling.

Polynucleotides of the present invention can also be used for genetherapy. Such polynucleotides can be introduced either in vivo or exvivo into cells for expression in a mammalian subject. Polynucleotidesof the invention may also be administered by other known methods forintroduction of nucleic acid into a cell or organism (including, withoutlimitation, in the form of viral vectors or naked DNA). Cells may alsobe cultured ex vivo in the presence of proteins of the present inventionin order to proliferate or to produce a desired effect on or activity insuch cells. Treated cells can then be introduced in vivo for therapeuticpurposes.

4.19.3 Effective Dosage

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein. For any compound used in the methodof the invention, the therapeutically effective dose can be estimatedinitially from appropriate in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat can be used to more accurately determine useful doses in humans.For example, a dose can be formulated in animal models to achieve acirculating concentration range that includes the IC₅₀ as determined incell culture (i.e., the concentration of the test compound whichachieves a half-maximal inhibition of the protein's biologicalactivity). Such information can be used to more accurately determineuseful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms or a prolongation of survivalin a patient. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds which exhibit high therapeutic indicesare preferred. The data obtained from these cell culture assays andanimal studies can be used in formulating a range of dosage for use inhuman. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. See, e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p.1. Dosage amount and interval may be adjusted individually toprovide plasma levels of the active moiety which are sufficient tomaintain the desired effects, or minimal effective concentration (MEC).The MEC will vary for each compound but can be estimated from in vitrodata. Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. However, HPLC assays orbioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%. In cases of local administration or selectiveuptake, the effective local concentration of the drug may not be relatedto plasma concentration.

An exemplary dosage regimen for polypeptides or other compositions ofthe invention will be in the range of about 0.01 μg/kg to 100 mg/kg ofbody weight daily, with the preferred dose being about 0.1 μg/kg to 25mg/kg of patient body weight daily, varying in adults and children.Dosing may be once daily, or equivalent doses may be delivered at longeror shorter intervals.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's age and weight, the severityof the affliction, the manner of administration and the judgment of theprescribing physician.

4.19.4 Packaging

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may, for example, comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. Compositions comprisinga compound of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabeled for treatment of an indicated condition.

4.20 Antibodies

Also included in the invention are antibodies to proteins, or fragmentsof proteins of the invention. The term “antibody” as used herein refersto immunoglobulin molecules and immunologically active portions ofimmunoglobulin (Ig) molecules, i.e., molecules that contain anantigen-binding site that specifically binds (immunoreacts with) anantigen. Such antibodies include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, F_(ab), F_(ab′) and F_((ab′)2)fragments, and an F_(ab) expression library. In general, an antibodymolecule obtained from humans relates to any of the classes IgG, IgM,IgA, IgE and IgD, which differ from one another by the nature of theheavy chain present in the molecule. Certain classes have subclasses aswell, such as IgG₁, IgG₂, and others. Furthermore, in humans, the lightchain may be a kappa chain or a lambda chain. Reference herein toantibodies includes a reference to all such classes, subclasses andtypes of human antibody species.

An isolated related protein of the invention may be intended to serve asan antigen, or a portion or fragment thereof, and additionally can beused as an immunogen to generate antibodies that immunospecifically bindthe antigen, using standard techniques for polyclonal and monoclonalantibody preparation. The full-length protein can be used or,alternatively, the invention provides antigenic peptide fragments of theantigen for use as immunogens. An antigenic peptide fragment comprisesat least 6 amino acid residues of the amino acid sequence of the fulllength protein, such as an amino acid sequence shown in SEQ ID NO: 5,7-13, 15, 17-24, 28, 30-156, 160, 162-182, 186, 188-213, 215, 217-239,241, 243-270, 272, 274-299, 302, 304-321, 323, 325-344, 348, 350-352,355, 357-376, 378, 380-401,408,410-414, 415, 420, 422-439,444-480,482-484, 487, 489-501, 505, 507-512, 516, 518-524, 528, 530-539,542, 544-546, 548, 550-553, 557, 559-567, 572, 574, 576, 579, 581-584,588, 590, 596, 602, 604-605, 607, 609-610, 612, 614-615, 618, 620, 622,624, 626, 628, 630, 632, or 634-653, or Tables 2-44 and encompasses anepitope thereof such that an antibody raised against the peptide forms aspecific immune complex with the full length protein or with anyfragment that contains the epitope. Preferably, the antigenic peptidecomprises at least 10 amino acid residues, or at least 15 amino acidresidues, or at least 20 amino acid residues, or at least 30 amino acidresidues. Preferred epitopes encompassed by the antigenic peptide areregions of the protein that are located on its surface; commonly theseare hydrophilic regions.

In certain embodiments of the invention, at least one epitopeencompassed by the antigenic peptide is a surface region of the protein,e.g., a hydrophilic region. A hydrophobicity analysis of the humanrelated protein sequence will indicate which regions of a relatedprotein are particularly hydrophilic and, therefore, are likely toencode surface residues useful for targeting antibody production. As ameans for targeting antibody production, hydropathy plots showingregions of hydrophilicity and hydrophobicity may be generated by anymethod well known in the art, including, for example, the Kyte Doolittleor the Hopp Woods methods, either with or without Fouriertransformation. See, e.g., Hopp and Woods, Proc. Nat. Acad. Sci. USA 78:3824-3828 (1981); Kyte and Doolittle, J. Mol. Biol. 157: 105-142 (1982),each of which is incorporated herein by reference in its entirety.Antibodies that are specific for one or more domains within an antigenicprotein, or derivatives, fragments, analogs or homologs thereof, arealso provided herein.

A protein of the invention, or a derivative, fragment, analog, homologor ortholog thereof, may be utilized as an immunogen in the generationof antibodies that immunospecifically bind these protein components.

The term “specific for” indicates that the variable regions of theantibodies of the invention recognize and bind polypeptides of theinvention exclusively (i.e., able to distinguish the polypeptide of theinvention from other similar polypeptides despite sequence identity,homology, or similarity found in the family of polypeptides), but mayalso interact with other proteins (for example, S. aureus protein A orother antibodies in ELISA techniques) through interactions withsequences outside the variable region of the antibodies, and inparticular, in the constant region of the molecule. Screening assays todetermine binding specificity of an antibody of the invention are wellknown and routinely practiced in the art. For a comprehensive discussionof such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual;Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter6. Antibodies that recognize and bind fragments of the polypeptides ofthe invention are also contemplated, provided that the antibodies arefirst and foremost specific for, as defined above, full-lengthpolypeptides of the invention. As with antibodies that are specific forfull length polypeptides of the invention, antibodies of the inventionthat recognize fragments are those which can distinguish polypeptidesfrom the same family of polypeptides despite inherent sequence identity,homology, or similarity found in the family of proteins.

Antibodies of the invention are useful for, for example, therapeuticpurposes (by modulating activity of a polypeptide of the invention),diagnostic purposes to detect or quantitate a polypeptide of theinvention, as well as purification of a polypeptide of the invention.Kits comprising an antibody of the invention for any of the purposesdescribed herein are also comprehended. In general, a kit of theinvention also includes a control antigen for which the antibody isimmunospecific. The invention further provides a hybridoma that producesan antibody according to the invention. Antibodies of the invention areuseful for detection and/or purification of the polypeptides of theinvention.

Monoclonal antibodies binding to the protein of the invention may beuseful diagnostic agents for the immunodetection of the protein.Neutralizing monoclonal antibodies binding to the protein may also beuseful therapeutics for both conditions associated with the protein andalso in the treatment of some forms of cancer where abnormal expressionof the protein is involved. In the case of cancerous cells or leukemiccells, neutralizing monoclonal antibodies against the protein may beuseful in detecting and preventing the metastatic spread of thecancerous cells, which may be mediated by the protein.

The labeled antibodies of the present invention can be used for invitro, in vivo, and in situ assays to identify cells or tissues in whicha fragment of the polypeptide of interest is expressed. The antibodiesmay also be used directly in therapies or other diagnostics. The presentinvention further provides the above-described antibodies immobilized ona solid support. Examples of such solid supports include plastics suchas polycarbonate, complex carbohydrates such as agarose and Sepharose®,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir, D. M. et al., “Handbook of Experimental Immunology” 4th Ed.,Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986);Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press, N.Y. (1974)). Theimmobilized antibodies of the present invention can be used for invitro, in vivo, and in situ assays as well as for immuno-affinitypurification of the proteins of the present invention.

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies directed against a protein of theinvention, or against derivatives, fragments, analogs homologs ororthologs thereof (see, for example, Antibodies: A Laboratory Manual,Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., incorporated herein by reference). Some of theseantibodies are discussed below.

4.20.1 Polyclonal Antibodies

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byone or more injections with the native protein, a synthetic variantthereof, or a derivative of the foregoing. An appropriate immunogenicpreparation can contain, for example, the naturally occurringimmunogenic protein, a chemically synthesized polypeptide representingthe immunogenic protein, or a recombinantly expressed immunogenicprotein. Furthermore, the protein may be conjugated to a second proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. The preparation can further include an adjuvant. Variousadjuvants used to increase the immunological response include, but arenot limited to, Freund's (complete and incomplete), mineral gels (e.g.,aluminum hydroxide), surface-active substances (e.g., lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol,etc.), adjuvants usable in humans such as Bacille Calmette-Guerin andCorynebacterium parvum, or similar immunostimulatory agents. Additionalexamples of adjuvants that can be employed include MPL-TDM adjuvant(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

The polyclonal antibody molecules directed against the immunogenicprotein can be isolated from the mammal (e.g., from the blood) andfurther purified by well known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

4.20.2 Monoclonal Antibodies

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs thus contain an antigen-binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes can beimmunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Preferably, antibodies having ahigh degree of specificity and a high binding affinity for the targetantigen are isolated.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.Suitable culture media for this purpose include, for example, Dulbecco'sModified Eagle's Medium and RPMI-1640 medium. Alternatively, thehybridoma cells can be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies can also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567; Morrison, Nature 368:812-13 (1994)) or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

4.20.3 Humanized Antibodies

The antibodies directed against the protein antigens of the inventioncan further comprise humanized antibodies or human antibodies. Theseantibodies are suitable for administration to humans without engenderingan immune response by the human against the administered immunoglobulin.Humanized forms of antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) that areprincipally comprised of the sequence of a human immunoglobulin, andcontain minimal sequence derived from a non-human immunoglobulin.Humanization can be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann, et al.,Nature, 332:323-327 (1988); Verhoeyen, et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. (See also U.S. Pat. No.5,225,539). In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies can also comprise residues that are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin consensussequence. The humanized antibody optimally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

4.20.4 Human Antibodies

Fully human antibodies relate to antibody molecules in which essentiallythe entire sequences of both the light chain and the heavy chain,including the CDRs, arise from human genes. Such antibodies are termed“human antibodies” or “fully human antibodies” herein. Human monoclonalantibodies can be prepared by the trioma technique; the human B-cellhybridoma technique (see Kozbor, et al., Immunol Today 4: 72 (1983)) andthe EBV hybridoma technique to produce human monoclonal antibodies (seeCole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized inthe practice of the present invention and may be produced by using humanhybridomas (see Cote, et al., Proc Natl Acad Sci USA 80: 2026-2030(1983)) or by transforming human B-cells with Epstein Barr Virus invitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96).

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)).Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.(Bio/Technology 10:779-783 (1992)); Lonberg et al. (Nature 368:856-859(1994)); Morrison (Nature 368:812-13 (1994)); Fishwild et al (NatureBiotechnology, 14:845-51 (1996)); Neuberger (Nature Biotechnology,14:826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13:65-93(1995)).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's WO94/02602). The endogenous genes encoding theheavy and light immunoglobulin chains in the nonhuman host have beenincapacitated, and active loci encoding human heavy and light chainimmunoglobulins are inserted into the host's genome. The human genes areincorporated, for example, using yeast artificial chromosomes containingthe requisite human DNA segments. An animal which provides all thedesired modifications is then obtained as progeny by crossbreedingintermediate transgenic animals containing fewer than the fullcomplement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed inPCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells which secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv molecules.

An example of a method of producing a nonhuman host, exemplified as amouse, lacking expression of an endogenous immunoglobulin heavy chain isdisclosed in U.S. Pat. No. 5,939,598. It can be obtained by a methodincluding deleting the J segment genes from at least one endogenousheavy chain locus in an embryonic stem cell to prevent rearrangement ofthe locus and to prevent formation of a transcript of a rearrangedimmunoglobulin heavy chain locus, the deletion being effected by atargeting vector containing a gene encoding a selectable marker; andproducing from the embryonic stem cell a transgenic mouse whose somaticand germ cells contain the gene encoding the selectable marker.

A method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771. It includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying aclinically relevant epitope on an immunogen, and a correlative methodfor selecting an antibody that binds immunospecifically to the relevantepitope with high affinity, are disclosed in PCT publication WO99/53049.

4.20.5 Fab Fragments and Single Chain Antibodies

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to an antigenic protein of theinvention (see e.g., U.S. Pat. No. 4,946,778). In addition, methods canbe adapted for the construction of F_(ab) expression libraries (seee.g., Huse, et al., Science 246:1275-1281 (1989)) to allow rapid andeffective identification of monoclonal F_(ab) fragments with the desiredspecificity for a protein or derivatives, fragments, analogs or homologsthereof. Antibody fragments that contain the idiotypes to a proteinantigen may be produced by techniques known in the art including, butnot limited to: (i) an F_((ab)2) fragment produced by pepsin digestionof an antibody molecule; (ii) an F_(ab) fragment generated by reducingthe disulfide bridges of an F_((ab′)2) fragment; (iii) an F_(ab)fragment generated by the treatment of the antibody molecule with papainand a reducing agent and (iv) F_(v) fragments.

4.20.6 Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is foran antigenic protein of the invention. The second binding target is anyother antigen, and advantageously is a cell-surface protein or receptoror receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are cotransfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full-length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148:1547-1553 (1992). Theleucine zipper peptides from the Fos and Jun proteins were linked to theFab′ portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. This method can also beutilized for the production of antibody homodimers. The “diabody”technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA90:6444-6448 (1993) has provided an alternative mechanism for makingbispecific antibody fragments. The fragments comprise a heavy-chainvariable domain (V_(H)) connected to a light-chain variable domain(V_(L)) by a linker which is too short to allow pairing between the twodomains on the same chain. Accordingly, the V_(H) and V_(L) domains ofone fragment are forced to pair with the complementary V_(L) and V_(H)domains of another fragment, thereby forming two antigen-binding sites.Another strategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See, Gruber et al.,J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in the protein antigen of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular antigen. Bispecific antibodies can alsobe used to direct cytotoxic agents to cells which express a particularantigen. These antibodies possess an antigen-binding arm and an armwhich binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interestbinds the protein antigen described herein and further binds tissuefactor (TF).

4.20.7 Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP03089). It is contemplated that the antibodies can be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins can beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

4.20.8 Effector Function Engineering

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedcan have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity can also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and can thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

4.20.9 Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody can be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is in turnconjugated to a cytotoxic agent.

4.21 Computer Readable Sequences

In one application of this embodiment, a nucleotide sequence of thepresent invention can be recorded on computer readable media. As usedherein, “computer readable media” refers to any medium which can be readand accessed directly by a computer. Such media include, but are notlimited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. A skilled artisan canreadily appreciate how any of the presently known computer readablemediums can be used to create a manufacture comprising computer readablemedium having recorded thereon a nucleotide sequence of the presentinvention. As used herein, “recorded” refers to a process for storinginformation on computer readable medium. A skilled artisan can readilyadopt any of the presently known methods for recording information oncomputer readable medium to generate manufactures comprising thenucleotide sequence information of the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon anucleotide sequence of the present invention. The choice of the datastorage structure will generally be based on the means chosen to accessthe stored information. In addition, a variety of data processorprograms and formats can be used to store the nucleotide sequenceinformation of the present invention on computer readable medium. Thesequence information can be represented in a word processing text file,formatted in commercially-available software such as WordPerfect andMicrosoft Word, or represented in the form of an ASCII file, stored in adatabase application, such as DB2, Sybase, Oracle, or the like. Askilled artisan can readily adapt any number of data processorstructuring formats (e.g. text file or database) in order to obtaincomputer readable medium having recorded thereon the nucleotide sequenceinformation of the present invention.

By providing any of the nucleotide sequences SEQ ID NO: 1-4, 6, 14, 16,25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273,300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407,409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517,526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578, 580, 587, 589,601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631or a representative fragment thereof; or a nucleotide sequence at least95% identical to any of the nucleotide sequences of SEQ ID NO: 1-4, 6,14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271,273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379,405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506,514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578,580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625,627, 629, or 631 in computer readable form, a skilled artisan canroutinely access the sequence information for a variety of purposes.Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable medium.The examples which follow demonstrate how software which implements theBLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE(Brutlag et al., Comp. Chem. 17:203-207 (1993)) search algorithms on aSybase system is used to identify open reading frames (ORFs) within anucleic acid sequence. Such ORFs may be protein-encoding fragments andmay be useful in producing commercially important proteins such asenzymes used in fermentation reactions and in the production ofcommercially useful metabolites.

As used herein, “a computer-based system” refers to the hardware means,software means, and data storage means used to analyze the nucleotidesequence information of the present invention. The minimum hardwaremeans of the computer-based systems of the present invention comprises acentral processing unit (CPU), input means, output means, and datastorage means. A skilled artisan can readily appreciate that any one ofthe currently available computer-based systems are suitable for use inthe present invention. As stated above, the computer-based systems ofthe present invention comprise a data storage means having storedtherein a nucleotide sequence of the present invention and the necessaryhardware means and software means for supporting and implementing asearch means. As used herein, “data storage means” refers to memorywhich can store nucleotide sequence information of the presentinvention, or a memory access means which can access manufactures havingrecorded thereon the nucleotide sequence information of the presentinvention.

As used herein, “search means” refers to one or more programs which areimplemented on the computer-based system to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of a known sequence which match a particular target sequence ortarget motif. A variety of known algorithms are disclosed publicly and avariety of commercially available software for conducting search meansare and can be used in the computer-based systems of the presentinvention. Examples of such software include, but are not limited to,Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). Askilled artisan can readily recognize that any one of the availablealgorithms or implementing software packages for conducting homologysearches can be adapted for use in the present computer-based systems.As used herein, a “target sequence” can be any nucleic acid or aminoacid sequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. The most preferred sequence length of atarget sequence is from about 10 to 100 amino acids, or from about 30 to300 nucleotide residues. However, it is well recognized that searchesfor commercially important fragments, such as sequence fragmentsinvolved in gene expression and protein processing, may be of shorterlength.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequence(s) are chosen based on a three-dimensional configurationwhich is formed upon the folding of the target motif There are a varietyof target motifs known in the art. Protein target motifs include, butare not limited to, enzyme active sites and signal sequences. Nucleicacid target motifs include, but are not limited to, promoter sequences,hairpin structures and inducible expression elements (protein bindingsequences).

4.22 Triple Helix Formation

In addition, the fragments of the present invention, as broadlydescribed, can be used to control gene expression through triple helixformation or antisense DNA or RNA, both of which methods are based onthe binding of a polynucleotide sequence to DNA or RNA. Polynucleotidessuitable for use in these methods are usually 20 to 40 bases in lengthand are designed to be complementary to a region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 15241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triplehelix-formation optimally results in a shut-off of RNA transcriptionfrom DNA, while antisense RNA hybridization blocks translation of anmRNA molecule into polypeptide. Both techniques have been demonstratedto be effective in model systems. Information contained in the sequencesof the present invention is necessary for the design of an antisense ortriple helix oligonucleotide.

4.23 Diagnostic Assays and Kits

The present invention further provides methods to identify the presenceor expression of one of the ORFs of the present invention, or homologthereof, in a test sample, using a nucleic acid probe or antibodies ofthe present invention, optionally conjugated or otherwise associatedwith a suitable label.

In general, methods for detecting a polynucleotide of the invention cancomprise contacting a sample with a compound that binds to and forms acomplex with the polynucleotide for a period sufficient to form thecomplex, and detecting the complex, so that if a complex is detected, apolynucleotide of the invention is detected in the sample. Such methodscan also comprise contacting a sample under stringent hybridizationconditions with nucleic acid primers that anneal to a polynucleotide ofthe invention under such conditions, and amplifying annealedpolynucleotides, so that if a polynucleotide is amplified, apolynucleotide of the invention is detected in the sample.

In general, methods for detecting a polypeptide of the invention cancomprise contacting a sample with a compound that binds to and forms acomplex with the polypeptide for a period sufficient to form thecomplex, and detecting the complex, so that if a complex is detected, apolypeptide of the invention is detected in the sample.

In detail, such methods comprise incubating a test sample with one ormore of the antibodies or one or more of the nucleic acid probes of thepresent invention and assaying for binding of the nucleic acid probes orantibodies to components within the test sample.

Conditions for incubating a nucleic acid probe or antibody with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid probe or antibody used in the assay. One skilled in the artwill recognize that any one of the commonly available hybridization,amplification or immunological assay formats can readily be adapted toemploy the nucleic acid probes or antibodies of the present invention.Examples of such assays can be found in Chard, T., An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985). The test samplesof the present invention include cells, protein or membrane extracts ofcells, or biological fluids such as sputum, blood, serum, plasma, orurine. The test sample used in the above-described method will varybased on the assay format, nature of the detection method and thetissues, cells or extracts used as the sample to be assayed. Methods forpreparing protein extracts or membrane extracts of cells are well knownin the art and can be readily be adapted in order to obtain a samplewhich is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention. Specifically, the invention provides a compartment kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the probes or antibodies of thepresent invention; and (b) one or more other containers comprising oneor more of the following: wash reagents, reagents capable of detectingpresence of a bound probe or antibody.

In detail, a compartment kit includes any kit in which reagents arecontained in separate containers. Such containers include small glasscontainers, plastic containers or strips of plastic or paper. Suchcontainers allows one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother. Such containers will include a container which will accept thetest sample, a container which contains the antibodies used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, etc.), and containers which contain thereagents used to detect the bound antibody or probe. Types of detectionreagents include labeled nucleic acid probes, labeled secondaryantibodies, or in the alternative, if the primary antibody is labeled,the enzymatic, or antibody binding reagents which are capable ofreacting with the labeled antibody. One skilled in the art will readilyrecognize that the disclosed probes and antibodies of the presentinvention can be readily incorporated into one of the established kitformats which are well known in the art.

4.24 Medical Imaging

The novel polypeptides and binding partners of the invention are usefulin medical imaging of sites expressing the molecules of the invention(e.g., where the polypeptide of the invention is involved in the immuneresponse, for imaging sites of inflammation or infection). See, e.g.,Kunkel et al., U.S. Pat. No. 5,413,778. Such methods involve chemicalattachment of a labeling or imaging agent, administration of the labeledpolypeptide to a subject in a pharmaceutically acceptable carrier, andimaging the labeled polypeptide in vivo at the target site.

4.25 Screening Assays

Using the isolated proteins and polynucleotides of the invention, thepresent invention further provides methods of obtaining and identifyingagents which bind to a polypeptide encoded by an ORF corresponding toany of the nucleotide sequences set forth in SEQ ID NO: 1-4, 6, 14, 16,25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273,300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407,409, 418-419,421,441-443, 485-486, 488, 503, 504, 506, 514-515, 517,526-527, 529, 547, 549, 556, 558, 570-571,573,577-578,580,587,589, 601,603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631, orbind to a specific domain of the polypeptide encoded by the nucleicacid. In detail, said method comprises the steps of:

-   -   (a) contacting an agent with an isolated protein encoded by an        ORF of the present invention, or nucleic acid of the invention;        and    -   (b) determining whether the agent binds to said protein or said        nucleic acid.

In general, therefore, such methods for identifying compounds that bindto a polynucleotide of the invention can comprise contacting a compoundwith a polynucleotide of the invention for a time sufficient to form apolynucleotide/compound complex, and detecting the complex, so that if apolynucleotide/compound complex is detected, a compound that binds to apolynucleotide of the invention is identified.

Likewise, in general, therefore, such methods for identifying compoundsthat bind to a polypeptide of the invention can comprise contacting acompound with a polypeptide of the invention for a time sufficient toform a polypeptide/compound complex, and detecting the complex, so thatif a polypeptide/compound complex is detected, a compound that binds toa polynucleotide of the invention is identified.

Methods for identifying compounds that bind to a polypeptide of theinvention can also comprise contacting a compound with a polypeptide ofthe invention in a cell for a time sufficient to form apolypeptide/compound complex, wherein the complex drives expression of areceptor gene sequence in the cell, and detecting the complex bydetecting reporter gene sequence expression, so that if apolypeptide/compound complex is detected, a compound that binds apolypeptide of the invention is identified.

Compounds identified via such methods can include compounds whichmodulate the activity of a polypeptide of the invention (that is,increase or decrease its activity, relative to activity observed in theabsence of the compound). Alternatively, compounds identified via suchmethods can include compounds which modulate the expression of apolynucleotide of the invention (that is, increase or decreaseexpression relative to expression levels observed in the absence of thecompound). Compounds, such as compounds identified via the methods ofthe invention, can be tested using standard assays well known to thoseof skill in the art for their ability to modulate activity/expression.

The agents screened in the above assay can be, but are not limited to,peptides, carbohydrates, vitamin derivatives, or other pharmaceuticalagents. The agents can be selected and screened at random or rationallyselected or designed using protein modeling techniques.

For random screening, agents such as peptides, carbohydrates,pharmaceutical agents and the like are selected at random and areassayed for their ability to bind to the protein encoded by the ORF ofthe present invention. Alternatively, agents may be rationally selectedor designed. As used herein, an agent is said to be “rationally selectedor designed” when the agent is chosen based on the configuration of theparticular protein. For example, one skilled in the art can readilyadapt currently available procedures to generate peptides,pharmaceutical agents and the like, capable of binding to a specificpeptide sequence, in order to generate rationally designed antipeptidepeptides, for example see Hurby et al., Application of SyntheticPeptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide,W.H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al, Biochemistry28:9230-8 (1989), or pharmaceutical agents, or the like.

In addition to the foregoing, one class of agents of the presentinvention, as broadly described, can be used to control gene expressionthrough binding to one of the ORFs or EMFs of the present invention. Asdescribed above, such agents can be randomly screened or rationallydesigned/selected. Targeting the ORF or EMF allows a skilled artisan todesign sequence specific or element specific agents, modulating theexpression of either a single ORF or multiple ORFs which rely on thesame EMF for expression control. One class of DNA binding agents areagents which contain base residues which hybridize or form a triplehelix formation by binding to DNA or RNA. Such agents can be based onthe classic phosphodiester, ribonucleic acid backbone, or can be avariety of sulfhydryl or polymeric derivatives which have baseattachment capacity.

Agents suitable for use in these methods usually contain 20 to 40 basesand are designed to be complementary to a region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triplehelix-formation optimally results in a shut-off of RNA transcriptionfrom DNA, while antisense RNA hybridization blocks translation of anmRNA molecule into polypeptide. Both techniques have been demonstratedto be effective in model systems. Information contained in the sequencesof the present invention is necessary for the design of an antisense ortriple helix oligonucleotide and other DNA binding agents.

Agents which bind to a protein encoded by one of the ORFs of the presentinvention can be used as a diagnostic agent. Agents which bind to aprotein encoded by one of the ORFs of the present invention can beformulated using known techniques to generate a pharmaceuticalcomposition.

4.26 Use of Nucleic Acids as Probes

Another aspect of the subject invention is to provide forpolypeptide-specific nucleic acid hybridization probes capable ofhybridizing with naturally occurring nucleotide sequences. Thehybridization probes of the subject invention may be derived from any ofthe nucleotide sequences SEQ ID NO: 1-4, 6, 14, 16, 25-27, 29, 157-159,161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324,345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421,441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547,549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606, 608,611, 613, 617, 619, 621, 623, 625, 627, 629, or 631. Because thecorresponding gene is only expressed in a limited number of tissues, ahybridization probe derived from of any of the nucleotide sequences SEQID NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161, 183-185, 187, 214, 216,240, 242, 271, 273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356,377, 379, 405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504,506, 514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571, 573,577-578, 580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621,623, 625, 627, 629, or 631 can be used as an indicator of the presenceof RNA of cell type of such a tissue in a sample.

Any suitable hybridization technique can be employed, such as, forexample, in situ hybridization. PCR as described in U.S. Pat. Nos.4,683,195 and 4,965,188 provides additional uses for oligonucleotidesbased upon the nucleotide sequences. Such probes used in PCR may be ofrecombinant origin, may be chemically synthesized, or a mixture of both.The probe will comprise a discrete nucleotide sequence for the detectionof identical sequences or a degenerate pool of possible sequences foridentification of closely related genomic sequences.

Other means for producing specific hybridization probes for nucleicacids include the cloning of nucleic acid sequences into vectors for theproduction of mRNA probes. Such vectors are known in the art and arecommercially available and may be used to synthesize RNA probes in vitroby means of the addition of the appropriate RNA polymerase as T7 or SP6RNA polymerase and the appropriate radioactively labeled nucleotides.The nucleotide sequences may be used to construct hybridization probesfor mapping their respective genomic sequences. The nucleotide sequenceprovided herein may be mapped to a chromosome or specific regions of achromosome using well known genetic and/or chromosomal mappingtechniques. These techniques include in situ hybridization, linkageanalysis against known chromosomal markers, hybridization screening withlibraries or flow-sorted chromosomal preparations specific to knownchromosomes, and the like. The technique of fluorescent in situhybridization of chromosome spreads has been described, among otherplaces, in Verma et al (1988) Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York N.Y.

Fluorescent in situ hybridization of chromosomal preparations and otherphysical chromosome mapping techniques may be correlated with additionalgenetic map data. Examples of genetic map data can be found in the 1994Genome Issue of Science (265:1981f). Correlation between the location ofa nucleic acid on a physical chromosomal map and a specific disease (orpredisposition to a specific disease) may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier or affected individuals.

4.27 Preparation of Support Bound Oligonucleotides

Oligonucleotides, i.e., small nucleic acid segments, may be readilyprepared by, for example, directly synthesizing the oligonucleotide bychemical means, as is commonly practiced using an automatedoligonucleotide synthesizer.

Support bound oligonucleotides may be prepared by any of the methodsknown to those of skill in the art using any suitable support such asglass, polystyrene or Teflon. One strategy is to precisely spotoligonucleotides synthesized by standard synthesizers. Immobilizationcan be achieved using passive adsorption (Inouye & Hondo, J. ClinMicrobiol 28:1462-72 (1990)); using UV light (Nagata et al., 1985;Dahlen et al., 1987; Morrissey & Collins, Mol. Cell Probes 3:189-207(1989)) or by covalent binding of base modified DNA (Keller et al.,1988; 1989); all references being specifically incorporated herein.

Another strategy that may be employed is the use of the strongbiotin-streptavidin interaction as a linker. For example, Broude et al.Proc. Natl. Acad. Sci USA 91:3072-6 (1994) describe the use ofbiotinylated probes, although these are duplex probes, that areimmobilized on streptavidin-coated magnetic beads. Streptavidin-coatedbeads may be purchased from Dynal, Oslo. Of course, this same linkingchemistry is applicable to coating any surface with streptavidin.Biotinylated probes may be purchased from various sources, such as,e.g., Operon Technologies (Alameda, Calif.).

Nunc Laboratories (Naperville, Ill.) is also selling suitable materialthat could be used. Nunc Laboratories have developed a method by whichDNA can be covalently bound to the microwell surface termed Covalink NH.CovaLink NH is a polystyrene surface grafted with secondary amino groups(>NH) that serve as bridge-heads for further covalent coupling. CovaLinkModules may be purchased from Nunc Laboratories. DNA molecules may bebound to CovaLink exclusively at the 5′-end by a phosphoramidate bond,allowing immobilization of more than 1 pmol of DNA (Rasmussen et al.,Anal Biochem 198:138-42 (1991)).

The use of CovaLink NH strips for covalent binding of DNA molecules atthe 5′-end has been described (Rasmussen et al., 1991). In thistechnology, a phosphoramidate bond is employed (Chu et al., NucleicAcids 11:6513-29 (1983)). This is beneficial as immobilization usingonly a single covalent bond is preferred. The phosphoramidate bond joinsthe DNA to the CovaLink NH secondary amino groups that are positioned atthe end of spacer arms covalently grafted onto the polystyrene surfacethrough a 2 nm long spacer arm. To link an oligonucleotide to CovaLinkNH via an phosphoramidate bond, the oligonucleotide terminus must have a5′-end phosphate group. It is, perhaps, even possible for biotin to becovalently bound to CovaLink and then streptavidin used to bind theprobes.

More specifically, the linkage method includes dissolving DNA in water(7.5 ng/ul) and denaturing for 10 min. at 95° C. and cooling on ice for10 min. Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm₇), is thenadded to a final concentration of 10 mM 1-MeIm₇. A ss DNA solution isthen dispensed into CovaLink NH strips (75 ul/well) standing on ice.

Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC),dissolved in 10 mM 1-MeIm₇, is made fresh and 25 ul added per well. Thestrips are incubated for 5 hours at 50° C. After incubation the stripsare washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3times, then they are soaked with washing solution for 5 min., andfinally they are washed 3 times (where in the washing solution is 0.4 NNaOH, 0.25% SDS heated to 50° C.).

It is contemplated that a further suitable method for use with thepresent invention is that described in PCT Patent Application WO90/03382 (Southern & Maskos), incorporated herein by reference. Thismethod of preparing an oligonucleotide bound to a support involvesattaching a nucleoside 3′-reagent through the phosphate group by acovalent phosphodiester link to aliphatic hydroxyl groups carried by thesupport. The oligonucleotide is then synthesized on the supportednucleoside and protecting groups removed from the syntheticoligonucleotide chain under standard conditions that do not cleave theoligonucleotide from the support. Suitable reagents include nucleosidephosphoramidite and nucleoside hydrogen phosphorate.

An on-chip strategy for the preparation of DNA probe for the preparationof DNA probe arrays may be employed. For example, addressablelaser-activated photodeprotection may be employed in the chemicalsynthesis of oligonucleotides directly on a glass surface, as describedby Fodor et al. Science 251:767-73 (1991)), incorporated herein byreference. Probes may also be immobilized on nylon supports as describedby Van Ness et al. Nucleic Acids Res. 19:3345-50 (1991); or linked toTeflon using the method of Duncan & Cavalier, Anal Biochem 169:104-8(1988); all references being specifically incorporated herein.

To link an oligonucleotide to a nylon support, as described by Van Nesset al. (1991), requires activation of the nylon surface via alkylationand selective activation of the 5′-amine of oligonucleotides withcyanuric chloride.

One particular way to prepare support bound oligonucleotides is toutilize the light-generated synthesis described by Pease et al., Proc.Natl. Acad. Sci USA 91:5022-6 (1994). These authors used currentphotolithographic techniques to generate arrays of immobilizedoligonucleotide probes (DNA chips). These methods, in which light isused to direct the synthesis of oligonucleotide probes in high-density,miniaturized arrays, utilize photolabile 5′-protectedN-acyl-deoxynucleoside phosphoramidites, surface linker chemistry andversatile combinatorial synthesis strategies. A matrix of 256 spatiallydefined oligonucleotide probes may be generated in this manner.

4.28 Preparation of Nucleic Acid Fragments

The nucleic acids may be obtained from any appropriate source, such ascDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands,cosmid or YAC inserts, and RNA, including mRNA without any amplificationsteps. For example, Sambrook et al. (1989) describes three protocols forthe isolation of high molecular weight DNA from mammalian cells (p.9.14-9.23).

DNA fragments may be prepared as clones in M13, plasmid or lambdavectors and/or prepared directly from genomic DNA or cDNA by PCR orother amplification methods. Samples may be prepared or dispensed inmultiwell plates. About 100-1000 ng of DNA samples may be prepared in2-500 ml of final volume.

The nucleic acids would then be fragmented by any of the methods knownto those of skill in the art including, for example, using restrictionenzymes as described at 9.24-9.28 of Sambrook et al. (1989), shearing byultrasound and NaOH treatment.

Low pressure shearing is also appropriate, as described by Schriefer etal. Nucleic Acids Res. 18:7455-6 (1990). In this method, DNA samples arepassed through a small French pressure cell at a variety of low tointermediate pressures. A lever device allows controlled application oflow to intermediate pressures to the cell. The results of these studiesindicate that low-pressure shearing is a useful alternative to sonic andenzymatic DNA fragmentation methods.

One particularly suitable way for fragmenting DNA is contemplated to bethat using the two base recognition endonuclease, CviJI, described byFitzgerald et al. Nucleic Acids Res. 20:3753-62 (1992). These authorsdescribed an approach for the rapid fragmentation and fractionation ofDNA into particular sizes that they contemplated to be suitable forshotgun cloning and sequencing.

The restriction endonuclease CviJI normally cleaves the recognitionsequence PuGCPy between the G and C to leave blunt ends. Atypicalreaction conditions, which alter the specificity of this enzyme(CviJI**), yield a quasi-random distribution of DNA fragments form thesmall molecule pUC19 (2688 base pairs). Fitzgerald et al. (1992)quantitatively evaluated the randomness of this fragmentation strategy,using a CviJI** digest of pUC19 that was size fractionated by a rapidgel filtration method and directly ligated, without end repair, to a lacZ minus M13 cloning vector. Sequence analysis of 76 clones showed thatCviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, andthat new sequence data is accumulated at a rate consistent with randomfragmentation.

As reported in the literature, advantages of this approach compared tosonication and agarose gel fractionation include: smaller amounts of DNAare required (0.2-0.5 μg instead of 2-5 μg); and fewer steps areinvolved (no preligation, end repair, chemical extraction, or agarosegel electrophoresis and elution are needed).

Irrespective of the manner in which the nucleic acid fragments areobtained or prepared, it is important to denature the DNA to give singlestranded pieces available for hybridization. This is achieved byincubating the DNA solution for 2-5 minutes at 80-90° C. The solution isthen cooled quickly to 2° C. to prevent renaturation of the DNAfragments before they are contacted with the chip. Phosphate groups mustalso be removed from genomic DNA by methods known in the art.

4.29 Preparation of DNA Arrays

Arrays may be prepared by spotting DNA samples on a support such as anylon membrane. Spotting may be performed by using arrays of metal pins(the positions of which correspond to an array of wells in a microtiterplate) to repeated by transfer of about 20 nl of a DNA solution to anylon membrane. By offset printing, a density of dots higher than thedensity of the wells is achieved. One to 25 dots may be accommodated in1 mm², depending on the type of label used. By avoiding spotting in somepreselected number of rows and columns, separate subsets (subarrays) maybe formed. Samples in one subarray may be the same genomic segment ofDNA (or the same gene) from different individuals, or may be different,overlapped genomic clones. Each of the subarrays may represent replicaspotting of the same samples. In one example, a selected gene segmentmay be amplified from 64 patients. For each patient, the amplified genesegment may be in one 96-well plate (all 96 wells containing the samesample). A plate for each of the 64 patients is prepared. By using a96-pin device, all samples may be spotted on one 8×12 cm membrane.Subarrays may contain 64 samples, one from each patient. Where the 96subarrays are identical, the dot span may be 1 mm² and there may be a 1mm space between subarrays.

Another approach is to use membranes or plates (available from NUNC,Naperville, Ill.) which may be partitioned by physical spacers e.g. aplastic grid molded over the membrane, the grid being similar to thesort of membrane applied to the bottom of multiwell plates, orhydrophobic strips. A fixed physical spacer is not preferred for imagingby exposure to flat phosphor-storage screens or x-ray films.

The present invention is illustrated in the following examples. Uponconsideration of the present disclosure, one of skill in the art willappreciate that many other embodiments and variations may be made in thescope of the present invention. Accordingly, it is intended that thebroader aspects of the present invention not be limited to thedisclosure of the following examples. The present invention is not to belimited in scope by the exemplified embodiments which are intended asillustrations of single aspects of the invention, and compositions andmethods which are functionally equivalent are within the scope of theinvention. Indeed, numerous modifications and variations in the practiceof the invention are expected to occur to those skilled in the art uponconsideration of the present preferred embodiments. Consequently, theonly limitations which should be placed upon the scope of the inventionare those which appear in the appended claims.

All references cited within the body of the instant specification arehereby incorporated by reference in their entirety.

5. EXAMPLES Example 1 Isolation Of SEQ ID NO: 1, 25, 157, 183, 300, 345,353, 405, 441, 486, 504, 515, 527, 571, 578, 630, and 632 from a cDNALibrary of Human Cells

The novel nucleic acids of SEQ ID NO: 1, 25, 157, 183, 300, 345, 353,405, 441, 486, 504, 515, 527, 571, 578, 630, and 632 were obtained fromvarious human cDNA libraries using standard PCR, sequencing byhybridization sequence signature analysis, and Sanger sequencingtechniques. The inserts of the library were amplified with PCR usingprimers specific for vector sequences flanking the inserts. Thesesamples were spotted onto nylon membranes and interrogated witholigonucleotide probes to give sequence signatures. The clones wereclustered into groups of similar or identical sequences, and singlerepresentative clones were selected from each group for gel sequencing.The 5′ sequence of the amplified inserts were then deduced using thereverse M13 sequencing primer in a typical Sanger sequencing protocol.PCR products were purified and subjected to fluorescent dye terminatorcycle sequencing. Single-pass gel sequencing was done using a 377Applied Biosystems (ABI) sequencer. These inserts were identified as anovel sequence not previously obtained from this library and notpreviously reported in public databases. These sequences are designatedas SEQ ID NO: 1, 25, 157, 183, 300, 345, 353, 405, 441, 486, 504, 515,527, 571, 578, 630, and 632 in the attached sequence listing.

Example 2 Assemblage of SEQ ID NO: 2, 3, 26, 158, 184, 299, or 346

The novel nucleic acids (SEQ ID NO: 2, 3, 26, 158, 184, 299, or 346) ofthe invention were assembled from sequences that were obtained fromvarious cDNA libraries by methods described in Example 1 above, and insome cases obtained from one or more public databases. The finalsequence was assembled using the EST sequence as seed. Then a recursivealgorithm was used to extend the seed into an extended assemblage, bypulling additional sequences from different databases (ie. Hyseq'sdatabase containing EST sequences, dbEST, gb pri, and UniGene) thatbelong to this assemblage. The algorithm terminated when there was noadditional sequences from the above databases that would extend theassemblage. Inclusion of component sequences into the assemblage wasbased on a BLASTN hit to the extending assemblage with BLAST scoregreater than 300 and percent identity greater than 95%.

The nearest neighbor results for the assembled contigs were obtained bya FASTA search against Genpept, using FASTXY algorithm. FASTXY is animproved version of FASTA alignment which allows in-codon frame shifts.The nearest neighbor results showed the closest homologue for eachassemblage from Genpept (and contain the translated amino acid sequencesfor which the assemblages encodes). The nearest neighbor results are setforth in Table 45 below: TABLE 45 Smith- SEQ ID Accession Waterman NO:No. Description Score % Identity 2 U27838 Mus musculus glycosyl- 41829.216 phosphatidyl-inositol- anchored protein homolog 3 M19419 Musmusculus proline-rich 244 36.683 salivary protein 158 X53556 Bos taurustype X collagen 657 42.963 184 L23982 Homo sapiens collagen 521 46.226type VII 346 AF095737 Homo sapiens unknown 366 68.085

The predicted amino acid sequences for SEQ ID NO: 2, 3, 26, 158, 184,299, or 346 was obtained by using a software program called FASTY(available from http://fasta:bioch.virginia.edu) which selects apolypeptide based on a comparison of translated novel polynucleotide toknown polynucleotides (W. R. Pearson, Methods in Enzymology, 183:63-98(1990), incorporated herein by reference). The results for SEQ ID NO: 2,3, 26, 158, 184, 299, or 346 are shown in Table 46 below, whereinA=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine,X=Unknown, *=Stop Codon, /=possible nucleotide deletion, \=possiblenucleotide insertion: TABLE 46 Predicted beginning Predicted endnucleotide nucleotide location location corresponding corresponding tofirst amino to first amino SEQ acid residue of acid residue of ID aminoacid amino acid Amino acid segment containing NO: sequence sequencesignal peptide 2 3 2456 FDTYRGLPSISNGNYSQLQFQAREYSGAPYSQRISMTTVSVAWKVLSGKIGEGAEGNCKCVISE GAWAVCPTQPCGKAKPDKHLKDLLSKLLNSGYFESIPVPKNAKEKEVPLEEEMLIQSEKKTQLS KTESVKESESLMEFAQPEIQPQEFLNRRYMTEVDYSNXQGEEQPWEADYARKPNLPKRWDML TEPDGQEKKQESFKSWEASGKHQEVSKPAVSLEQRKQDTSKLRSTLPEEQKKQEISKSKPSPSQ WKQDTPKSKAGYVQEEHKKQETPKLWPVQLQKEQDPKKQTPKSWTPSMQSEQNTTKSWTTP MCEEQDSKQPETPKSWENNVESQKIHSLTSQSQISPKSWGVATASLWNDQLLPRKLNTEPKDVP /IACASA*GFLPLQPPFRRI/HVLRKEKLQDLMTQIQGTCNFMQESVLDFDKPSSAIPTSQPPSATP G*PRRHLKEQNLS\VKVLFFQGAVT\VFNVNAPLPPRKEQEIKIESPYSPGYNQSFTTASTQTPPQC QLPSIIHVEQTVHSQETANYHPDGTIQVSNGSLAFYPAQTNVFPRPTQPFVNSRGSVRGCTRGGR LITNSYRSPGGYKGFDTYRGLPSISNGNYSQLQFQAREYSGAPYSQRDNFQQCYKRGGTSGGPR ANSRAGWSDSSQVSSPERDNETFNSGDSGQGDSRSMTPVDVPVTNPAATILPVHVYPLPQQMR VAFSAARTSNLAPGTLDQPEVFDLLLNNLGETFDLQLGRFNCPVNGTYVFIFHMLKLAVNVIPLY VNLMKNEEVLVSAYANDGAPDHETASNHAILQLFQGDQIWLRLHRGAIYGSSW (SEQ ID NO: 23) 3 39 599GASPNQGQNRPHARQRAPPQ/G/PPGEPERRAP LPSGHGEPCRHRPPPFPQPP/AGTQKPLLQGPGGG*PAENAPTAALGSPAPPRGCQAAPPPRSGA GRPDLPTLAGPRPAPA\PPPSAAPPPPPSGAPSR/PAAGRQRLSGVSSGPSLGWW*VGRGRGLPAF AQIAGHQVGPRRRRTPAGRKPRSPAGPR (SEQ ID NO:24) 26 202 2471 FDSAVLSSINVMAVLPGPLQLLGVLLTISLSSIRLIQAGAYYGIIKPLPPQIPPQMPPQIPQYQPLGQ QVPHMPLAKDGLAMGKEMPHLQYGKEYPHLPQYMIKEIQPAPRMGKEAVPKKGKEIPLASLRG EQGPRGEPGPRGPPGPPGLPGHGIPGIXGKPGPQGYPGVGKPGMPGMPGKPGAMGMPGAKGEI GQKGEIGPMGLP*PQGPPGPHGLPGIGKPGGPGLPGQPGPKGDRGPKGLPGPQGLRGPKGDKGF GMPGAPGVKGPPGMHGPPGPVGLPGVGKPGVTGFPGP\QGPLGK\PGAPGEPGPQGPIGVPGVQ GPPGIPGIGKPGQDG\IPGQPGFPGGKGEQGLPGLPGPPGLPGIGKPGFPGPKGDRGMGGVPGAL GPRGEKGPIGAPGIGGPPGEPGLPGIPGPMGPPGAIGFPGPKGEGGIVGPQGPPGPKGEPGLQGFP GKPGFLGEVGPPGMRGFPGPIGPKGEHGQKGVPGLPGVPGLLGPKGEPGIPGDQGLQGPPGWG IGGPSGPIGPPGIPGPKGEPGLPGPPGFPGIGKPGVAGLHGPPGKPGALGPQGQPGLPGPPGPPGP PGPPAVMPPTPPPQGEYLPDMGLGIDGVKPPHAYGAKKGKNGGPAYEMPAFTABLTAPFPPVG APVKFNKLLYNGRQNYNPQTGIFTCEVPGVYYFAYHVHCKGGNVWVALFKNNEPVMYTYDE YKKGFLDQASGSAVLLLRPGDRVFLQMPSEQAAGLYAGQYVHSSFSGYLLYPM (SEQ ID NO: 156) 158 142 1058SSKTPAVGRSCEQEPKMFVLLYVTSFAICASG QPRGNQLKGENYSPRYICSLPGLPGPPGPPGANGSPGPHGRIGLPGRDGRDGRKGEKGEKGTAG LRGKTGPLGLAGEKGDQGETGKKGPIGPEGEKGEVGPIGPPGPKGDRGEQGDPGLPGVCRCGS IVLKSAFSVGITTSYPEERLPIIFNKVLFNEGEHYNPATGKFICAFPGIYYFSYDITLANKHLAIGL VHNGQYRIKTFDANTGNHDVASGSTVIYLQPEDEVWLEIFFTDQNGLFSDPGWADSLFSGFLLY VDTDYLDSISEDDEL (SEQ ID NO: 182) 184739 794 ASFLLQMCP*GPVQSLSSEP*GSGGFCLPLKSAQGT*T/PQDTCRQGHPGLPGNPGHNGLPGRDG RDGAKGDKGDAGEPGRPGSPGKDGTSGEKGERGADGKVEAKGIKGDQGS\*GSPGKHGPKGLA GPMGEKGLRGETGPQGQKGNXGDVGPTGPEGPRGNIGPLGPTGLPGPMGPIGKPGPKGEAGPT GPQGEPGVRGIRGWKGDRGEKGKIGETLVLPKSAFTVGLTVLSKFPSSDVPIKFDKIHIT (SEQ ID NO: 299) 346 2471 2985ETSLERERLSFCTGSRTTRSAELKAVGFEAALQ EVITPEVVPASQSEAYQTLRQNQAQVHNFFFFWGGDSPTLSPRLECSSMSAHCNLRLPGSSNSP TSASRVAGTTGACRHARLIFCILVEMGFHRVAQAGRELLSSANPPTSASQSAGITGMSHHAQPS SQLLISSCC (SEQ ID NO: 352)

Example 3 Assemblage of SEQ ID NO: 4, 14, 27, 159, 185, 214, 240, or 271

The novel nucleic acids (SEQ ID NO: 4, 14, 27, 159, 185, 214, 240, or271) of the invention were assembled from sequences that were obtainedfrom cDNA libraries by methods described in Example 1 above, and in somecases obtained from one or more public databases. The final sequenceswere assembled using the EST sequences as seed. Then a recursivealgorithm was used to extend the seed into an extended assemblage, bypulling additional sequences from different databases (i.e. Hyseq'sdatabase containing EST sequences, dbEST, gb pri, and UniGene) thatbelong to this assemblage. The algorithm terminated when there was noadditional sequences from the above databases that would extend theassemblage. Inclusion of component sequences into the assemblage wasbased on a BLASTN hit to the extending assemblage with BLAST scoregreater than 300 and percent identity greater than 95%.

Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full-length genecDNA sequence and its corresponding protein sequence were generated fromthe assemblage. Any frame shifts and incorrect sop codons were correctedby hand editing. During editing, the sequence was checked using FASTYand BLAST against Genbank (i.e. dbEST, gb pri, UniGene, Genpept). Othercomputer programs which may have been used in the editing process werephredPhrap and Consed (University of Washington) and ed-ready, ed-extand cg-zip-2 (Hyseq, Inc.). The full-length nucleotide sequences areshown in the Sequence Listing as SEQ ID NO: 4, 14, 27, 159, 185, 214,240, or 271; and the full-length amino acid sequences are shown in thesequence listing as SEQ ID NO: 5, 15, 28, 160, 186, 215, 241, or 272.

Further annotation of SEQ ID NO: 4, or 14 can be found in U.S. patentapplication Ser. No. 09/598,075 filed Jun. 20, 2000 (attorney docket no.787); herein incorporated by reference in its entirety.

Further annotation of SEQ ID NO: 27 can be found in U.S. patentapplication Ser. No. 09/620,312 filed Jul. 19, 2000 (attorney docket no.784); herein incorporated by reference in its entirety.

Further annotation of SEQ ID NO: 159 can be found in U.S. patentapplication Ser. No. 09/728,952 filed Nov. 30, 2000 (attorney docket no.799); herein incorporated by reference in its entirety.

Further annotation of SEQ ID NO: SEQ ID NO: 185, 214, 240, or 271 can befound in U.S. Provisional patent application Ser. No. 60/306,971 filedJul. 21, 2001 (attorney docket no. 805); herein incorporated byreference in its entirety.

Example 4 Assemblage of SEQ ID NO: 301, 322, 347, 354, or 377

The novel nucleic acid (SEQ ID NO: 301, 322, 347, 354, or 377) of theinvention were assembled from sequences that was obtained from a cDNAlibrary by methods described in Example 1 above, and in some casesobtained from one or more public databases. The final sequence wasassembled using the EST sequences as seed. Then a recursive algorithmwas used to extend the seed into an extended assemblage, by pullingadditional sequences from different databases (i.e. Hyseq's databasecontaining EST sequences, dbEST, gb pri, and UniGene) that belong tothis assemblage. The algorithm terminated when there was no additionalsequences from the above databases that would extend the assemblage.Inclusion of component sequences into the assemblage was based on aBLASTN hit to the extending assemblage with BLAST score greater than 300and percent identity greater than 95%.

Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full-length genecDNA sequence and its corresponding protein sequence were generated fromthe assemblage. Any frame shifts and incorrect sop codons were correctedby hand editing. During editing, the sequence was checked using FASTYand BLAST against Genbank (ie. dbEST, gb pri, UniGene, Genpept). Othercomputer programs which may have been used in the editing process werephredPhrap and Consed (University of Washington) and ed-ready, ed-extand cg-zip-2 (Hyseq, Inc.). The full-length nucleotide sequences areshown in the Sequence Listing as SEQ ID NO: 301, 322, 347, 354, or 377;and the full-length amino acid sequences are shown in the sequencelisting as SEQ ID NO: 302, 323, 348, 355, or 378.

Example 5 Assemblage of SEQ ID NO: 406

The novel nucleic acid (SEQ ID NO: 406) of the invention was assembledfrom sequences that were obtained from various cDNA libraries by methodsdescribed in Example 1 above, and in some cases obtained from one ormore public databases. The final sequence was assembled using the ESTsequence as seed. Then a recursive algorithm was used to extend the seedinto an extended assemblage, by pulling additional sequences fromdifferent databases (i.e. Hyseq's database containing EST sequences,dbEST, gb pri, and UniGene) that belong to this assemblage. Thealgorithm terminated when there was no additional sequences from theabove databases that would extend the assemblage. Inclusion of componentsequences into the assemblage was based on a BLASTN hit to the extendingassemblage with BLAST score greater than 300 and percent identitygreater than 95%.

The nearest neighbor result for the assembled contigs were obtained by aFASTA search against Genpept, using FASTXY algorithm. FASTXY is animproved version of FASTA alignment which allows in-codon frame shifts.The nearest neighbor result showed the closest homologue for eachassemblage from Genpept (and contains the translated amino acidsequences for which the assemblage encodes). The nearest neighbor resultis set forth in Table 47 below: TABLE 47 Smith- SEQ ID AccessionWaterman NO: No. Description Score % Identity 406 X89015 Homo sapiensleupin 996 44.162

The predicted amino acid sequences for SEQ ID NO: 406 were obtained byusing a software program called FASTY (available fromhttp://fasta:bioch.virginia.edu) which selects a polypeptide based on acomparison of translated novel polynucleotide to known polynucleotides(W. R. Pearson, Methods in Enzymology, 183:63-98 (1990), incorporatedherein by reference). The results for SEQ ID NO: 406 are shown in Table48 below wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine,X=Unknown, *=Stop Codon, /=possible nucleotide deletion, \=possiblenucleotide insertion: TABLE 48 Predicted beginning Predicted endnucleotide nucleotide location location corresponding corresponding tofirst amino to first amino acid residue of acid residue of amino acidamino acid Amino acid segment containing sequence sequence signalpeptide 1 851 MRASPLEGNSGKNTDSSNITQKPELVPPDLWTHPERLATPQQTPTELQESFASTLETTSLISNNLL LKMSQSKATVEGLKEAKLGWSLSPGPTHLVLTEPHQ1VISFTMDSLVTANTKFCFDLFQEIGKD DRHKNIFFSPLSLSAALGMVRLGARSDSAHQIDEVLHFNEFSQNESKEPDPCLKSNKQKVLADS SLEGQKKTTEPLDQQAGSLNNESGLVSCYFGQLLSKLDRIKTDYTLSIANRLYGEQEFPICQEYL DGVIQFYHTTIESVDFQKNPEKSRQEINFWVECQSQGKIKELFSKDAINAIETVLVLVNAVYFKAK WETYFDHENTVDAPFWLNANENKSVKMMTQKGLYRJGFIEEVKAQILEMRYTKGKLSMFVLL PSHSKDNLKGLEELERKITYEKMVAWSSSENMSEESVVLSFPRFTLEDSYDLNSILQDMGITDI FDETRADLTGISPSPNLYLSKIIHKTFVEVDENGTQAAAATGAVVSESSKNSHLWLAPFMHPAQ AGVKRSAAGIVDGWPPYAPLSAFWPPECSAMTTDTSNSHILFGVSLFPLELPPVVQGGHAVFLQK AGLEQTKEMALFSIRDEIDTDVSLELLTAFEESCQLHVA (SEQ ID NO: 415)

Example 6 Assemblage of SEQ ID NO: 407

The novel nucleic acid (SEQ ID NO: 407) of the invention were assembledfrom sequences that was obtained from a cDNA library by methodsdescribed in Example 1 above, and in some cases obtained from one ormore public databases. The final sequence was assembled using the ESTsequences as seed. Then a recursive algorithm was used to extend theseed into an extended assemblage, by pulling additional sequences fromdifferent databases (i.e. Hyseq's database containing EST sequences,dbEST, gb pri, and UniGene) that belong to this assemblage. Thealgorithm terminated when there was no additional sequences from theabove databases that would extend the assemblage. Inclusion of componentsequences into the assemblage was based on a BLASTN hit to the extendingassemblage with BLAST score greater than 300 and percent identitygreater than 95%.

Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full-length genecDNA sequence and its corresponding protein sequence were generated fromthe assemblage. Any frame shifts and incorrect sop codons were correctedby hand editing. During editing, the sequence was checked using FASTYand BLAST against Genbank (i.e. dbEST, gb pri, UniGene, Genpept). Othercomputer programs which may have been used in the editing process werephredPhrap and Consed (University of Washington) and ed-ready, ed-extand cg-zip-2 (Hyseq, Inc.).

Example 7 Identification of SEQ ID NO: 419

Assembly of the novel nucleotide sequence of SEQ ID NO: 419 wasaccomplished using a contig sequence SEQ ID NO: 418 as a seed. The seedwas extended by gel sequencing (377 Applied Biosystems (ABI) sequencer)using primers to extend the 3′ end (primer extension). The DNA from thefull-length clone was then isolated, sonicated and recloned for gelsequencing. Each fragment was sequenced by gel sequencing (377 AppliedBiosystems (ABI) sequencer) and the sequences were assembled to arriveat the complete sequence.

A polypeptide (SEQ ID NO: 420) was predicted to be encoded by SEQ ID NO:419 as set forth below. The polypeptide was predicted using a softwareprogram called BLASTX which selects a polypeptide based on a comparisonof the translated novel polynucleotide to known polynucleotides. Theinitial methionine starts at position 1217 of SEQ ID NO: 419 and theputative stop codon, TGA, begins at position 2414 of the nucleotidesequence SEQ ID NO: 419.

Example 8 Assemblage of SEQ ID NO: 442

A polypeptide was predicted to be encoded by SEQ ID NO: 442 as set forthbelow. The polypeptide was predicted using a software program calledFASTY (available from http://fasta.bioch.virginia.edu) which selects apolypeptide based on a comparison of translated novel polynucleotide toknown polypeptides (W. R. Pearson, Methods in Enzymology, 183:63-98(1990), herein incorporated by reference). The results for SEQ ID NO:442 are shown in Table 49 below, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine, X=Unknown, *=Stop Codon, /=possiblenucleotide deletion, \=possible nucleotide insertion: TABLE 49 PredictedPredicted beginning end nucleotide nucleotide location locationcorrespond- correspond- ing to first ing to last amino acid amino acidresidue of residue of amino acid amino acid Amino acid segmentcontaining segment segment signal peptide 3 385PPGPKGDQGDEGKEGRPGIPGLPGLRGLPGERGT PGLPGPKGNDGKLGATGPMGMRGFKGDRGPKGEKGEKGDRAGDASGVEAPMMIRLVNGSGPHEG RVEVYHDRRWGTVCDDGWDKXDGDVVCRM (SEQ IDNO: 480)

Example 9 Assemblage of SEQ ID NO: 443 and 444

Assembly of novel nucleotide sequence of SEQ ID NO: 443 was accomplishedby using an EST sequence SEQ ID NO: 441 as a seed. The seed was extendedby gel sequencing (377 Applied Biosystems (ABI) sequencer) using primersto extend the 3′ end (primer extension). A portion of the 5′ end wasextended by using primers and 5′ RACE on a reverse transcribed cDNAmixture prepared from mRNAs from Invitrogen that included adult brain,kidney, heart, liver, lung, placenta, small intestine, and uterus, aswell as fetal brain, heart, kidney, liver, lung, muscle, and skin; mRNAsfrom Clontech that included adult adrenal gland, bone marrow, lymphnode, pituitary gland, spinal cord, spleen, thyroid gland, thymus, andtrachea, as well as the MOLT-4 leukemia cell line; and mRNAs fromBiochain that included adult esophagus, and fetal umbilical cord. Theresulting sequence was used to conduct a BLASTN alignment againstGENSCAN (Stanford University, Burge, C. and Karlin, S. (1997) Predictionof complete gene structures in human genomic DNA. J. Mol. Biol. 268,78-94) predicted genes from the Human Genome Project database to arriveat the final 5′ complete DNA sequence.

A polypeptide (SEQ ID NO: 444) was predicted to be encoded by SEQ ID NO:443 as set forth below. The polypeptide was predicted using a softwareprogram called BLASTX which selects a polypeptide based on a comparisonof translated novel polynucleotide to known polynucleotides. The initialmethionine starts at position 417 of SEQ ID NO: 443 and the putativestop codon, TGA, begins at position 1902 of the nucleotide sequence.

Example 10 Assemblage of SEQ ID NO: 486, 504, 515, or 527

The novel nucleic acids (SEQ ID NO: 486, 504, 515, or 527) of theinvention were assembled from sequences that were obtained from variouscDNA libraries by methods described in Example 1 above, and in somecases obtained from one or more public databases. The final sequence wasassembled using the EST sequence as seed. Then a recursive algorithm wasused to extend the seed into an extended assemblage, by pullingadditional sequences from different databases (i.e. Hyseq's databasecontaining EST sequences, dbEST, gb pri, and UniGene) that belong tothis assemblage. The algorithm terminated when there was no additionalsequences from the above databases that would extend the assemblage.Inclusion of component sequences into the assemblage was based on aBLASTN hit to the extending assemblage with BLAST score greater than 300and percent identity greater than 95%.

The nearest neighbor results for the assembled contigs were obtained bya FASTA search against Genpept, using FASTXY algorithm. FASTXY is animproved version of FASTA alignment which allows in-codon frame shifts.The nearest neighbor results showed the closest homologue for eachassemblage from Genpept (and contain the translated amino acid sequencesfor which the assemblages encodes). The nearest neighbor results are setforth in Table 50 below: TABLE 50 Smith- SEQ ID Accession Waterman NO:No. Description Score % Identity 486 AAW29667 Homo sapiens DL185_1 396960.629 clone secreted protein 504 AK009118 Mus musculus 1089 74.797putativeprotein 515 AB052620 Mus musculus DDM36 1374 40.830 527 U35371Rattus norvegicus neural 2822 91.391 cell adhesion protein precursorBIG-2

The predicted amino acid sequences for SEQ ID NO: 486, 504, 515, or 527were predicted as set forth below. The polypeptides were predicted usinga software program called BLASTX which selects a polypeptide based on acomparison of the translated novel polynucleotide to knownpolynucleotides. The initial methionine of SEQ ID NO: 487 starts atposition 178 of SEQ ID NO: 486 and the putative stop codon, TGA, beginsat position 3262 of the nucleotide sequence SEQ ID NO: 486. The initialmethionine of SEQ ID NO: 506 starts at position 17 of SEQ ID NO: 504 andthe putative stop codon, TGA, begins at position 707 of the nucleotidesequence SEQ ID NO: 504. The initial methionine of SEQ ID NO: 516 startsat position 1 of SEQ ID NO: 505 and the putative stop codon TAG, beginsat position 2000 of the nucleotide sequence SEQ ID NO: 505. The initialmethionine of SEQ ID NO: 528 starts at position 117 of SEQ ID NO: 527and the putative stop codon TGA, begins at position 3249 of thenucleotide sequence SEQ ID NO: 527.

Example 11 Assemblage of SEQ ID NO: 547 or 556

The novel nucleic acids (SEQ ID NO: 547 or 556) of the invention wereassembled from sequences that were obtained from cDNA libraries bymethods described in Example 1 above, and in some cases obtained fromone or more public databases. The final sequences were assembled usingthe EST sequences as seed. Then a recursive algorithm was used to extendthe seed into an extended assemblage, by pulling additional sequencesfrom different databases (i.e. Hyseq's database containing ESTsequences, dbEST, gb pri, and UniGene) that belong to this assemblage.The algorithm terminated when there was no additional sequences from theabove databases that would extend the assemblage. Inclusion of componentsequences into the assemblage was based on a BLASTN hit to the extendingassemblage with BLAST score greater than 300 and percent identitygreater than 95%.

Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full-length genecDNA sequence and its corresponding protein sequence were generated fromthe assemblage. Any frame shifts and incorrect sop codons were correctedby hand editing. During editing, the sequence was checked using FASTYand BLAST against Genbank (i.e. dbEST, gb pri, UniGene, Genpept). Othercomputer programs which may have been used in the editing process werephredphrap and Consed (University of Washington) and ed-ready, ed-extand cg-zip-2 (Hyseq, Inc.). The full-length nucleotide sequences areshown in the Sequence Listing as SEQ ID NO: 547 or 556; and thefull-length amino acid sequences are shown in the sequence listing asSEQ ID NO: 548 or 557.

Example 12 Assemblage of SEQ ID NO: 571 or 578

The novel nucleic acids (SEQ ID NO: 571 or 578) of the invention wereassembled from sequences that were obtained from various cDNA librariesby methods described in Example 1 above, and in some cases obtained fromone or more public databases. The final sequence was assembled using theEST sequence as seed. Then a recursive algorithm was used to extend theseed into an extended assemblage, by pulling additional sequences fromdifferent databases (i.e. Hyseq's database containing EST sequences,dbEST, gb pri, and UniGene) that belong to this assemblage. Thealgorithm terminated when there was no additional sequences from theabove databases that would extend the assemblage. Inclusion of componentsequences into the assemblage was based on a BLASTN hit to the extendingassemblage with BLAST score greater than 300 and percent identitygreater than 95%.

The nearest neighbor results for the assembled contigs were obtained bya FASTA search against Genpept, using FASTXY algorithm. FASTXY is animproved version of FASTA alignment which allows in-codon frame shifts.The nearest neighbor results showed the closest homologue for eachassemblage from Genpept (and contain the translated amino acid sequencesfor which the assemblages encodes). The nearest neighbor results are setforth in Table 51 below: TABLE 51 Smith- SEQ ID Accession Waterman % NO:No. Description Score Identity 573 X83006 Homo sapiens neutrophils 20840 gelatinase associated lipocalin 578 AAY91653 Human secreted protein606 100 sequence encoded by gene 62 SEQ ID NO 326

The predicted amino acid sequences for SEQ ID NO: 571 or 578 werepredicted as set forth below. The polypeptides were predicted using asoftware program called BLASTX which selects a polypeptide based on acomparison of the translated novel polynucleotide to knownpolynucleotides. The initial methionine of SEQ ID NO: 572 starts atposition 192 of SEQ ID NO: 571 and the putative stop codon, TGA, beginsat position 660 of the nucleotide sequence SEQ ID NO: 571. The initialmethionine of SEQ ID NO: 579 starts at position 128 of SEQ ID NO: 578and the putative stop codon, TGA, begins at position 727 of thenucleotide sequence SEQ ID NO: 578.

Example 13 Assemblage of SEQ ID NO: 587 and 589

The novel nucleic acid (SEQ ID NO: 587 and 589) of the invention wereassembled from sequences that was obtained from a cDNA library bymethods described in Example 1 above, and in some cases obtained fromone or more public databases. The final sequence was assembled using theEST sequences as seed. Then a recursive algorithm was used to extend theseed into an extended assemblage, by pulling additional sequences fromdifferent databases (ie. Hyseq's database containing EST sequences,dbEST, gb pri, and UniGene) that belong to this assemblage. Thealgorithm terminated when there was no additional sequences from theabove databases that would extend the assemblage. Inclusion of componentsequences into the assemblage was based on a BLASTN hit to the extendingassemblage with BLAST score greater than 300 and percent identitygreater than 95%.

Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full-length genecDNA sequence and its corresponding protein sequence were generated fromthe assemblage. Any frame shifts and incorrect stop codons werecorrected by hand editing. During editing, the sequence was checkedusing FASTY and BLAST against Genbank (i.e. dbEST0627_Hs, gb pri124_Hs,UniGene124, Genpept124). Other computer programs which may have beenused in the editing process were phredPhrap and Consed (University ofWashington) and ed-ready, ed-ext and cg-zip-2 (Hyseq, Inc.). Thefull-length nucleotide sequences are shown in the Sequence Listing asSEQ ID NO: 587 and 589; and the full-length amino acid sequences areshown in the sequence listing as SEQ ID NO: 587.

Example 14 Assemblage of SEQ ID NO: 601, 606, and 611

The novel nucleic acids (SEQ ID NO: 601, 606, and 611) of the inventionwere assembled from sequences that were obtained from cDNA libraries bymethods described in Example 1 above, and in some cases obtained fromone or more public databases. The final sequences were assembled usingthe EST sequences as seed. Then a recursive algorithm was used to extendthe seed into an extended assemblage, by pulling additional sequencesfrom different databases (i.e. Hyseq's database containing ESTsequences, dbEST, gb pri, and UniGene) that belong to this assemblage.The algorithm terminated when there was no additional sequences from theabove databases that would extend the assemblage. Inclusion of componentsequences into the assemblage was based on a BLASTN hit to the extendingassemblage with BLAST score greater than 300 and percent identitygreater than 95%.

Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full-length genecDNA sequence and its corresponding protein sequence were generated fromthe assemblage. Any frame shifts and incorrect sop codons were correctedby hand editing. During editing, the sequence was checked using FASTYand BLAST against Genbank (i.e. dbEST, gb pri, UniGene, Genpept). Othercomputer programs which may have been used in the editing process werephredPhrap and Consed (University of Washington) and ed-ready, ed-extand cg-zip-2 (Hyseq, Inc.). The full-length nucleotide sequences areshown in the Sequence Listing as SEQ ID NO: 601, 606, and 611; and thefull-length amino acid sequences are shown in the sequence listing asSEQ ID NO: 602, 607, and 612.

Example 15 Assemblage of SEQ ID NO: 629 and 631

The novel nucleic acid (SEQ ID NO: 629 and 631) of the invention wereassembled from sequences that was obtained from a cDNA library bymethods described in Example 1 above, and in some cases obtained fromone or more public databases. The final sequence was assembled using theEST sequences as seed. Then a recursive algorithm was used to extend theseed into an extended assemblage, by pulling additional sequences fromdifferent databases (i.e. Hyseq's database containing ESTsequences013001, dbEST013001 HS, gb pri126_HS_cd, and UniGene126) thatbelong to this assemblage. The algorithm terminated when there was noadditional sequences from the above databases that would extend theassemblage. Inclusion of component sequences into the assemblage wasbased on a BLASTN hit to the extending assemblage with BLAST scoregreater than 300 and percent identity greater than 95%.

Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a fill-length genecDNA sequence and its corresponding protein sequence were generated fromthe assemblage. Any frame shifts and incorrect sop codons were correctedby hand editing. During editing, the sequence was checked using FASTYand BLAST against Genbank (i.e. dbEST013001_HS, gb pri126_HS_cd,UniGene126, Genpept127). Other computer programs which may have beenused in the editing process were phredPhrap and Consed (University ofWashington) and ed-ready, ed-ext and cg-zip-2 (Hyseq, Inc.). Thefull-length nucleotide sequences are shown in the Sequence Listing asSEQ ID NO: 629 and 631; and the full-length amino acid sequences areshown in the sequence listing as SEQ ID NO: 630 and 632.

Example 16 Tissue Expression Analysis and Chromosomal Location of SEQ IDNO: 4, 14, 27, 159, 186, 214, 240, 271, 301, 322, 347, 354, or 377

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 4, or 14 is found to be expressed in followinghuman tissue/cell cDNA (see Table 52): TABLE 52 Library No. of PositiveTotal No. of Clones Name Clones in the Library Tissue Origin BMD001 13342599 bone marrow ABD003 3 83268 adult brain FLS001 30 555770 fetalliver-spleen AKD001 5 176438 adult kidney LUC001 5 210372 leukocytesATS001 2 26744 testis AKT002 7 149669 adult kidney AOV001 22 259409adult ovary IB2002 21 265743 infant brain LGT002 7 158948 lung tumorHFB001 5 74494 fetal brain IBS001 3 33191 infant brain LPC001 8 97546lymphocyte PIT004 5 120274 pituitary gland SPC001 2 61905 whole organTHM001 4 113947 thymus THR001 2 124110 thyroid gland ADR002 5 90185adrenal gland CVX001 7 125473 cervix THA002 1 32817 thalamus FUC001 1125570 umbilical cord SIN001 2 142562 whole organ ABR001 3 30163 adultbrain FLG001 2 28154 whole organ BLD001 3 29386 bladder FSK001 5 127263fetal skin CLN001 3 28708 colon REC001 1 28337 rectum SPLc01 2 110573spleen FLG003 1 27360 fetal lung NTU001 4 37055 neuronal cells NTD001 535080 induced neuronal cells NTR001 3 34629 retinoic acid-inducedneuronal cells ABR006 1 108204 adult brain FBR004 1 27560 fetal brainFBR006 8 151893 fetal brain ABR008 14 145661 adult brain FLS002 58709733 fetal liver-spleen IB2003 14 201294 infant brain ADP001 2 37287cultured preadipocytes ADP002 1 32855 cultured preadipocytes FLV002 232865 fetal liver BMD002 1 75816 bone marrow DIA002 1 40119 diaphragmFLV004 3 74491 fetal liver FKD002 1 33111 fetal kidney FSK002 1 72628fetal skin FLS003 9 187791 fetal liver-spleen HMP001 3 71425 macrophageFLG004 1 41090 fetal lung BMD008 1 44770 bone marrow DGD001 1 91971lymphocyte DGD004 1 91423 lymphocytes STM001 2 181899 bone marrow OBE013 132217 adipocytes

SEQ ID NO: 4, or 14 were further analyzed for their presence in thepublic dbEST database and their tissue source. SEQ ID NO: 4 or 14 werefound to be expressed in following tissues: Gessler Wilms tumor, colon,Stratagene hNT neuron, Fibroblasts, senescent, Stratagene endothelialcell 937223, Soares breast 2NbHBst, Stratagene lung carcinoma 937218,Soares fetal liver spleen 1NFLS, Soares_parathyroid_tumor_NbHPA, totalbrain, Soares_NhHMPu_S1, Soares_fetal_heart_NbHH19W, liver, Soaresinfant brain 1NIB, Jurkat T-cells, cochlea, Ovary, and Testis tumor.

The gene corresponding to SEQ ID NO: 4 or 14 was mapped to humanchromosome 12p11-37.2 by BLAST analysis with human genome sequences.

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 27 is found to be expressed in following humantissue/cell cDNA (see Table 53): TABLE 53 Total No. of No. of PositiveClones in the Library Name Clones Library Tissue Origin LGT002 5 158948lung tumor MMG001 1 131991 mammary gland PIT004 1 120274 pituitary glandTHR001 5 124110 thyroid gland ADR002 2 90185 adrenal gland TRC001 123820 trachea FUC001 17 125570 umbilical cord FLG001 1 28154 whole organFSK001 1 127263 fetal skin ADP001 1 37287 adipocytes ADP002 7 32855adipocytes PLA003 1 80877 placenta FKD002 1 33111 fetal kidney FSK002 172628 fetal skin FHR001 2 108446 fetal heart FLG004 1 41090 fetal lungOBE01 5 132217 adipocytes

SEQ ID NO: 27 was further analyzed for their presence in the publicdbEST database and their tissue source. SEQ D NO: 27 was found to beexpressed in following tissues: Bone, poorly differentiated adeno,Fibroblasts, senescent, melanocyte, colon tumor RER+, Soares_NhHu_S1,bone marrow stroma, 2 pooled tumors (clear cell, Soares ovary tumorNbHOT, cochlea.

The gene corresponding to SEQ ID NO: 27 was mapped to chromosome 3 byBLAST analysis with human genome sequences.

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 159 is found to be expressed in followinghuman tissue/cell cDNA (see Table 54): TABLE 54 Total No. of No. ofPositive Clones in the Library Name Clones Library Tissue Origin FLS0011 555770 fetal liver-spleen AKD001 3 176438 adult kidney AOV001 9 259409adult ovary CVX001 2 125473 adult cervix FLG001 1 28154 fetal lungSPLc01 1 110573 spleen FKD002 2 33111 fetal kidney

SEQ ID NO: 159 was further analyzed for their presence in the publicdbEST database and their tissue source. SEQ ID NO: 159 was found to beexpressed in following tissues: Soares_NhHMPu_S1, NCI_CGAP_Sub6.

The gene corresponding to SEQ ID NO: 159 was mapped to human chromosome4 by BLAST analysis with human genome sequences.

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 186, 214, 240, or 271 is found to be expressedin following human tissue/cell cDNA (see Table 55): TABLE 55 Total No.of No. of Positive Clones in the Library Name Clones Library TissueOrigin FLS001 1 555770 fetal liver-spleen FMS001 1 32743 Fetal muscleFSK001 1 127263 Fetal skin FMS002 6 40223 Fetal muscle FHR001 4 108446Fetal heart

SEQ ID NO: 186, 214, 240, or 271 was further analyzed for their presencein the public dbEST database and their tissue source. SEQ ID NO: 186,214, 240, or 271 was found to be expressed in following tissues:

-   HEMBB1, head_normal, MAGE resequences, MAGM, bone marrow, larynx    tumor, high grade preneoplastic lesion, NCI_CGAP_Sub7,    NIH_MGC_(—)87, NIH_MGC_(—)91, Soares_NFL_T_GBC_S1,    Soares_testis_NHT.

The gene corresponding to SEQ ID NO: 186, 214, 240, or 271 was mapped tohuman chromosome 13 by BLAST analysis with human genome sequences.

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 301, or 322 is found to be expressed infollowing human tissue/cell cDNA (see Table 56): TABLE 56 Total No. ofNo. of Positive Clones in the Library Name Clones Library Tissue OriginFLS001 1 555770 Fetal liver-spleen LUC001 1 210372 leukocytes AKT002 1149669 adult kidney IB2002 2 265743 infant brain HFB001 3 74494 fetalbrain SPC001 1 61905 whole organ NTR001 1 34629 retinoic acid-inducedneuronal cells STM001 1 181899 bone marrow

SEQ ID NO: 301, or 322 was further analyzed for their presence in thepublic dbEST database and their tissue source. SEQ ID NO: 301, or 322was found to be expressed in following tissues: 2 pooled tumors, HTC,and Soares fetal liver spleen 1NFLS S1.

The gene corresponding to SEQ ID NO: 301 or 322 was mapped to humanchromosome 18 by BLAST analysis with human genome sequences.

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 347 is found to be expressed in followinghuman tissue/cell cDNA (see Table 57): TABLE 57 Total No. of No. ofPositive Clones in the Library Name Clones Library Tissue Origin BMD0012 342599 bone marrow ABD003 16 83268 adult brain FLS001 2 555770 fetalliver-spleen AKD001 2 176438 adult kidney LUC001 3 210372 leukocytesLUC003 3 30296 leukocytes ALV001 1 30866 young liver ATS001 1 26744testis ASP001 1 32114 adult spleen APL001 1 31936 placenta ABT004 73231910 adult brain AKT002 2 149669 adult kidney ALV002 10 144402 adultliver AOV001 5 259409 ovary IB2002 1276 265743 infant brain LGT002 16158948 adult lung MMG001 8 131991 mammary gland HFB001 38 74494 fetalbrain FBT002 1 35745 fetal brain IBM002 99 13952 infant brain IBS001 18233191 infant brain LPC001 3 97546 lymphocyte PIT004 3 120274 pituitarygland SPC001 1705 61905 whole organ THR001 1 124110 thyroid gland MEL00417 30503 melanoma ADR002 3 90185 adrenal gland CVX001 4 125473 cervixPRT001 2 28649 whole organ THA002 591 32817 thalamus TRC001 1 23820trachea FBR001 1 28664 fetal brain FUC001 8 125570 umbilical cord SKM0011 28327 whole organ SIN001 6 142562 whole organ ABR001 241 30163 adultbrain FLG001 2 28154 whole organ BLD001 43 29386 bladder FMS001 4 32743fetal muscle FSK001 8 127263 fetal skin CLN001 4 28708 colon REC001 328337 rectum SPLc01 13 110573 spleen FLG003 8 27360 fetal lung THMc02 1796791 thymus NTU001 2 37055 neuronal cells NTR001 2 34629 retinoicacid-induced neuronal cells ABR006 365 108204 adult brain FBR004 2 27560fetal brain FBR006 351 151893 fetal brain ABR008 11420 145661 adultbrain FLS002 4 709733 fetal liver-spleen IB2003 1108 201294 infant brainADP001 2 37287 cultured preadipocytes FLV002 11 32865 fetal liver PLA0032 80877 placenta FLV004 2 74491 fetal liver ESO002 2 36840 esophagusFSK002 4 72628 fetal skin FMS002 7 40223 fetal muscle FHR001 7 108446fetal heart FLS003 4 187791 fetal liver-spleen HMP001 10 71425macrophage FLG004 1 41090 fetal lung ABR016 57 45716 brain BMD008 344770 bone marrow LYN001 2 44025 lymph node STM001 3 181899 bone marrow

SEQ ID NO: 347 was further analyzed for their presence in the publicdbEST database and their tissue source. SEQ ID NO: 347 was found to beexpressed in following tissues: Soares_total_fetus_Nb2HF8_(—)9w,head_neck, kidney tumor, colon tumor RER+, Soares_fetal_heart_NbHH19W,head_neck, pooled germ cell tumors, kidney, subtracted, 2 pooled tumors(clear cell type), colon tumor RER+, malignant melanoma, metastatic tolymph node, LTI_NFL006_PL2, cervix carcinoma cell line, bone marrow cellline, melanotic melanoma, carcinoid, Pineal gland II.

The gene corresponding to SEQ ID NO: 347 was mapped to human chromosome18p11.3 by BLAST analysis with human genome sequences.

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 354, or 377 is found to be expressed infollowing human tissue/cell cDNA (see Table 58): TABLE 58 Total No. ofNo. of Positive Clones in the Library Name Clones Library Tissue OriginALV002 1 144402 adult liver FBR006 1 151893 fetal brain FKD002 1 33111fetal kidney FSK002 1 72628 fetal skin

SEQ ID NO: 354 or 377 was further analyzed for their presence in thepublic dbEST database and their tissue source. SEQ ID NO: 354 or 377 wasfound to be expressed in following tissues: Neuroblastoma cells.

The gene corresponding to SEQ D NO: 354 or 377 was mapped to chromosome12 by BLAST analysis with human genome sequences.

Example 17 Chromosomal Localization of SEQ ID NO: 240

To determine the chromosomal localization of SEQ ID NO: 240, genespecific PCR primers (5′-AAGCCTGGTCCCAAAGGAGA-3′ and5′-GGTGTGGCGGATTTTTAAACTCT-3′) were screened against the NIGMShuman/rodent somatic cell hybrid mapping panel #2. PCR amplification ofthe 423 nt product was performed using the following conditions; aninitial denaturation at 94° C. for 3 min, followed by 5 cycles of 30 sat 94° C., 30 sec at 68° C. and 1 min at 72° C., followed by 5 cycles of30 s at 94° C., 30 sec at 64° C. and 1 min at 72° C., followed by 20cycles of 30 s at 94° C., 30 sec at 60° C. and 1 min at 72° C. followedby an extension of 10 min at 72° C. All products were separated by 3%agarose gel electrophoresis and visualized via ethidium bromidestaining. SEQ ID NO: 240 was mapped to chromosome 13.

Example 18 Tissue Expression Analysis and Chromosomal Localization of407

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 407 was found to be expressed in followinghuman tissue/cell cDNA (see Table 59): TABLE 59 No. of Positive TotalNo. of Clones Library Name Clones in the Library Tissue Origin FSK001 1127263 fetal skin FSK002 2 72628 fetal skin

SEQ ID NO: 407 was further analyzed for their presence in the publicdbEST database and their tissue source. SEQ ID NO: 407 was found to beexpressed in following tissues: Soares_NhHMPu_S1.

The gene corresponding to SEQ ID NO: 407 was mapped to human chromosome18 by BLAST analysis with human genome sequences.

Example 19 Tissue Expression Analysis and Chromosomal Localization of486, 504 and 527

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 486, is found to be expressed in followinghuman tissue/cell cDNA (see Table 60): TABLE 60 No. of Positive TotalNo. of Clones Library Name Clones in the Library Tissue Origin AKD001 1176438 Adult kidney HFB001 1 74494 Fetal brain PIT004 2 120274 Pituitarygland SPC001 1 61905 Spinal cord FUC001 1 125570 Umbilical cord SIN001 1142562 Small intestine FBR004 1 27560 Fetal brain IB2003 1 201294 Infantbrain

SEQ ID NO: 486 was further analyzed for their presence in the publicdbEST database and their tissue source. SEQ ID NO: 486 was found to beexpressed in following tissues: pituitary, NIH_MGC_(—)114 adult brain,NIH_MGC_(—)121 fetal brain, Soares multiple sclerosis lesions,hypothalamus, Athersys RAGE library, Clontech human aorta polyA+ mRNA,NCI_CGAP_Kid11 subtracted kidney, Morton fetal cochlea, NIH_MGC_(—)120adult pancreas spleen, Soares_fetal_lung_NbHL19W,NCI_CGAP_Utlwell-differentiated endometrial adenocarcinoma, 7 pooledtumors, NT, and NCI_CGAP_Pr28 subtracted prostate.

The gene corresponding to SEQ ID NO: 486 was mapped to human chromosome3p by BLAST analysis with human genome sequences.

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 504 is found to be expressed in followinghuman tissue/cell cDNA (see Table 61): TABLE 61 Total No. of No. ofPositive Clones in the Library Name Clones Library Tissue Origin FKD0021 33111 Fetal kidney

SEQ ID NO: 504 was further analyzed for its presence in the public dbESTdatabase and its tissue source. SEQ ID NO: 504 was found to be expressedin following tissues: normal colon and adult colon kidney stomach.

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 514 is found to be expressed in followinghuman tissue/cell cDNA (see Table 62): TABLE 62 Total No. of No. ofPositive Clones in the Library Name Clones Library Tissue Origin FKD0011 127263 Fetal skin

SEQ ID NO: 514 was further analyzed for its presence in the public dbESTdatabase and its tissue source. SEQ ID NO: 514 was found to be expressedin following tissues: Soares_NFL_T_G testis, B-cell and fetal lung.

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 527 is found to be expressed in followinghuman tissue/cell cDNA (see Table 63): TABLE 63 Total No. of No. ofPositive Clones in the Library Name Clones Library Tissue Origin PIT0041 120274 Pituitary gland THM001 1 113947 Thymus CVX001 1 125473 CervixSIN001 1 142562 Whole organ IB2003 1 201294 Infant brain

SEQ ID NO: 527 was further analyzed for its presence in the public dbESTdatabase and its tissue source. SEQ ID NO: 527 was found to be expressedin following tissues: normal nervous, testis, NIH_MGC_(—)85 lymph,lymphoma, NIH_MGC_(—)97 testis cell line, NCI_CGAP_Skn3 skin, Schilleroligodendroglioma, and Soares_NFL_T_G tesis, B-cell and fetal lung.

Example 20 Tissue Expression of 547

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 547 is found to be expressed in the followinghuman tissue/cell cDNA and shown in Table 64: TABLE 64 Total No. ofClones in the No. of Positive Library Name Tissue Origin Library ClonesAPL001 placenta 31936 3963 PIT004 pituitary gland 120274 10496 PLA003placenta 80877 4991 FLS001 fetal liver-spleen 555770 8220 FLS003 fetalliver-spleen 187791 2072 FLV001 fetal liver 33189 46 ALN001 lymph node27965 17 ABT004 adult brain 31910 19 FKD001 fetal kidney 31293 16 FLS002fetal liver-spleen 709733 211 LUC003 leukocytes 30296 8 OBE01adipocytes/Obesity 132217 31 FBR004 fetal brain 27560 6 BMD001 bonemarrow 342599 56 ADP002 adipocytes 32855 4 THM001 thymus 113947 10THMc02 thymus 96791 8 FSK002 fetal skin 72628 5 ASP001 adult spleen32114 2 THR001 thyroid gland 124110 7 DGD004 lymphocytes/Meyloma 91423 5FLV004 fetal liver 74491 4 ADP001 adipocytes 37287 2 AKD001 adult kidney176438 9 FMS002 fetal muscle 40223 2 FUC001 umbilical cord 125570 6AHR001 adult heart 130524 6 ABR016 brain 45716 2 BMD002 bone marrow75816 3 AOV001 ovary 259409 10 ABD003 adult brain 83268 3 BLD001 bladder29386 1 ABR001 adult brain 30163 1 DGD001 lymphocyte/Burkitt's 91971 3lymphoma ALV001 young liver 30866 1 SPC001 whole organ 61905 2 THA002thalamus 32817 1 NTR001 neuron 34629 1 NTD001 neuron 35080 1 ABR006adult brain 108204 3 MMG001 mammary gland 131991 3 ADR002 adrenal gland90185 2 STM001 bone marrow 181899 4 LPC001 lymphocyte 97546 2 IB2003infant brain 201294 4 FBR006 fetal brain 151893 3 CVX001 cervix 125473 2FSK001 fetal skin 127263 2 BB2002 infant brain 265743 4 ABR008 adultbrain 145661 2 HFB001 fetal brain 74494 1 AKT002 adult kidney 149669 2LUC001 leukocytes 210372 2 SIN001 whole organ 142562 1 ALV002 adultliver 144402 1 LGT002 lung tumor 158948 1 EDT001 endothelial cells177809 1

Example 21 Tissue Expression Analysis of 571 and 578

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 571 and 578 are found to be expressed infollowing human tissue/cell cDNA (see Table 65): TABLE 65 No. ofPositive Total No. of Clones Library Name Clones in the Library TissueOrigin AKT002 2 149669 Adult kidney THR001 2 124110 Thyroid gland CVX0011 125473 Cervix EDT001 1 177809 Endothelial cells SPLc01 1 110573(Spleen) THMc02 1 96791 Thymus ABR006 7 108204 Adult brain ABR008 1145661 Adult brain ALV003 1 34611 Adult liver FLV002 1 32865 Fetal liverPLA003 1 80877 Placenta FSK002 1 72628 Fetal skin FMS002 1 40223 Fetalmuscle FHR001 2 108446 Fetal heart SIP002 17 179333 Mixed tissue SIP0052 37621 Mixed tissue

Example 22 Tissue Expression Analysis of 587 and 589

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 587 or 589 is found to be expressed infollowing human tissue/cell cDNA (see Table 66). TABLE 66 LibraryLibrary Number of Total number Name clones clones Tissue 71 ADR002 190185 Adrenal gland 118 SPLc01 1 110573 spleen

Expression of SEQ ID 587 and 589 was also found in the lung tumorlibrary (LGT002).

SEQ ID NO: 587 was further analyzed for its presence in the public dbESTdatabase and its tissue source. SEQ ID NO: 587 was found to be expressedin following tissues: T colon, Soares_placenta_(—)8 to 9 weeks_(—)2NbHP8to 9W, NIH_MGC_(—)90 Liver tumor cell line made from adenocarcinomatissue and Soares_NFL_T_G_testis, B-cell and fetal lung. Furtherinformation on these libraries may be obtained athttp://image.llnl.gov/image/html/humlib_info.shtml.

Example 23 Chromosomal Localization of SEQ ID NO: 587

By running Hyseq's proprietary software program that maps SEQ ID NO: 587to the human genome, SEQ ID NO: 587 was mapped to chromosome1p22.2-31.1. To confirm the chromosomal localization of SEQ ID NO: 587,gene specific PCR primers (5′ATGGCACATCGTGATTCTGAG 3 and5′-TTAGCAGAACTTTAGC-3′) were screened against the NIGMS human/rodentsomatic cell hybrid mapping panel #2. PCR amplification of the 423 ntproduct was performed using the following conditions; an initialdenaturation at 94° C. for 3 min, followed by 5 cycles of 30 s at 94°C., 30 sec at 68° C. and 1 min at 72° C., followed by 5 cycles of 30 sat 94° C., 30 sec at 64° C. and 1 min at 72° C., followed by 20 cyclesof 30 s at 94° C., 30 sec at 60° C. and 1 min at 72° C. followed by anextension of 10 min at 72° C. All products were separated by 3% agarosegel electrophoresis and visualized via ethidium bromide staining. SEQ IDNO: 587 was mapped to chromosome 1.

Example 24 Tissue Expression Analysis of 601, 606, and 611

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 601 is found to be expressed in the followinghuman tissue/cell cDNA (see Table 67): TABLE 67 Total No. of Clones inthe No. of Positive Library Name Tissue Origin Library Clones SIN001whole organ 142562 3802 BMD002 bone marrow 75816 714 SPLc01 spleen110573 356 STO001 whole organ 26894 307 REC001 rectum 28337 244 TRC001trachea 23820 235 BMD001 bone marrow 342599 213 SAL001 whole organ 37753152 CLN001 colon 28708 92 CLN001 colon 28708 92 CVX001 cervix 125473 85THM001 thymus 113947 51 THMc02 thymus 96791 38 THR001 Thyroid gland124110 27 PRT001 whole organ 28649 22 LPC001 lymphocyte 97546 20 ASP001adult spleen 32114 18 AKT002 adult kidney 149669 17 FLG001 whole organ28154 17 BLD001 bladder 29386 15 ESO002 esophagus 36840 12 ABR016 brain45716 11 LUC001 leukocytes 210372 10 FUC001 umbilical cord 125570 10UTR001 uterus 29595 7 PIT004 pituitary gland 120274 6 PIT004 pituitarygland 120274 6 FSK001 fetal skin 127263 4 FLS002 fetal liver-spleen709733 4 ALG001 adult lung 28271 3 HFB001 fetal brain 74494 3 SPC001whole organ 61905 3 HMP001 marrow 71425 3 FLS001 fetal liver spleen555770 2 ALV002 adult liver 144402 2 LGT002 lung tumor 158948 2 ABR006adult brain 108204 2 PLA003 placenta 80877 2 AHR001 adult heart 130524 1MMG001 mammary gland 131991 1 ADR002 adrenal gland 90185 1 THA002thalamus 32817 1 SKM001 whole organ 28327 1 NTD001 neuron 35080 1 FBR004fetal brain 27560 1 FBR006 fetal brain 151893 1 ALV003 adult liver 346111 FKD002 fetal kidney 33111 1 FSK002 fetal liver-spleen 187791 1

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 606 and 611 are found to be identicallyexpressed in the following human tissue/cell cDNA (see Table 68): TABLE68 Total No. of Clones in the No. of Positive Library Name Tissue OriginLibrary Clones SIN001 whole organ 142562 3802 BMD002 bone marrow 75816714 SPLc01 spleen 110573 356 THMc02 thymus 96791 338 STO001 whole organ26894 307 REC001 rectum 28337 244 TRC001 trachea 23820 235 BMD001 bonemarrow 342599 213 SAL001 whole organ 37753 152 LYN001 lymph node 44025112 BMD008 bone marrow 44770 104 CLN001 colon 28708 92 CVX001 cervix125473 85 THM001 thymus 113947 51 THR001 thyroid gland 124110 27 PRT001whole organ 28649 22 LPC001 lymphocyte 97546 20 ASP001 adult spleen32114 18 AKT002 adult kidney 149669 17 FLG001 fetal lung 28154 17 BLD001bladder 29386 15 ESO002 esophagus 36840 12 ABR016 brain 45716 11 LUC001leukocytes 210372 10 FUC001 umbilical cord 125570 10 UTR001 uterus 295957 PIT004 pituitary gland 120274 6 FSK002 fetal skin 72628 5 STM001 bonemarrow 181899 4 DGD004 lymphocytes 91423 4 FLS002 fetal liver-spleen709733 4 FSK001 fetal skin 127263 4 SPC001 whole organ 61905 3 HFB001fetal brain 74494 3 HMP001 hematopoetic cells 71425 3 ALG001 adult lung28271 3 FLS001 fetal liver-spleen 555770 2 LGT002 lung tumor 158948 2ALV002 adult liver 144402 2 FHR001 fetal heart 108446 2 PLA003 placenta80877 2 ABR006 adult brain 108204 2 SKM001 whole organ 28327 1 THA002thalamus 32817 1 ADR002 adrenal gland 90185 1 DGD001 lymphocyte 91971 1MMG001 mammary gland 131991 1 AHR001 adult heart 130524 1 FLS003 fetalliver-spleen 187791 1 FKD002 fetal kidney 33111 1 ALV003 adult liver34611 1 FBR006 fetal brain 151893 1 FBR004 fetal brain 27560 1 NTD001neuron 35080 1

Example 25 Tissue Expression Analysis and Chromosomal Location of SEQ IDNO: 629

By checking Hyseq proprietary database established from screening byhybridization, SEQ ID NO: 629 is found to be expressed in followinghuman tissue/cell cDNA (see Table 69): TABLE 69 No. of Positive TotalNo. of Clones Library Name Clones in the Library Tissue Origin BMD001145 342599 bone marrow ABD003 96 83268 adult brain FLS001 506 555770fetal liver-spleen AKD001 421 176438 adult kidney LUC001 245 210372leukocytes LUC003 31 30296 leukocytes ALV001 10 30866 young liver ATS00134 26744 testis ASP001 78 32114 adult spleen ALG001 35 28271 adult lungAHR001 328 130524 adult heart APL001 6 31936 placenta FKD001 34 31293fetal kidney ALN001 34 27965 lymph node ABT004 4 31910 adult brainAKT002 50 149669 adult kidney ALV002 24 144402 adult liver AOV001 124259409 ovary IB2002 4 265743 infant brain LGT002 59 158948 lung tumorMMG001 82 131991 mammary gland AB3001 1 1565 adult brain HFB001 30 74494fetal brain FLV001 11 33189 fetal liver FBT002 8 35745 fetal brainLPC001 17 97546 lymphocyte PIT004 70 120274 pituitary gland SPC001 3761905 whole organ THM001 56 113947 thymus THR001 85 124110 thyroid glandMEL004 24 30503 melanoma ADR002 41 90185 adrenal gland CVX001 81 125473cervix PRT001 19 28649 whole organ STO001 18 26894 whole organ THA002 1232817 thalamus TRC001 20 23820 trachea UTR001 7 29595 uterus FBR001 928664 fetal brain FUC001 46 125570 umbilical cord SKM001 13 28327 wholeorgan SAL001 10 37753 whole organ SIN001 55 142562 whole organ ABR001 1330163 adult brain FLG001 12 28154 whole organ BLD001 8 29386 bladderFMS001 17 32743 fetal muscle FSK001 27 127263 fetal skin EDT001 30177809 endothelial cells CLN001 2 28708 colon REC001 2 28337 rectumSPLc01 8 110573 adult spleen FLG003 3 27360 fetal lung THMc02 3 96791thymus NTU001 8 37055 neuronal cells NTD001 3 35080 neuron NTR001 734629 neuron ABR006 3 108204 adult brain FBR004 3 27560 fetal brainFBR006 7 151893 fetal brain ABR008 1 145661 adult brain FLS002 52 709733fetal liver-spleen IB2003 1 201294 infant brain ADP001 14 37287adipocytes ADP002 10 32855 adipocytes LFB001 1 41616 lung, fibroblastALV003 10 34611 adult liver FLV002 13 32865 fetal liver BMD002 35 75816bone marrow DIA002 4 40119 diaphragm PLA003 2 80877 placenta FLV004 574491 fetal liver FKD002 7 33111 fetal kidney ESO002 6 36840 esophagusFSK002 5 72628 fetal skin FMS002 11 40223 fetal muscle FHR001 22 108446fetal heart FLS003 171 187791 fetal liver-spleen HMP001 13 71425macrophage FLG004 14 41090 fetal lung ABR016 3 45716 brain SUP002 28179333 mix 16 tissues SUP005 1 37621 mix 16 tissues SUP007 14 43646 mix16 tissues BMD008 12 44770 bone marrow LYN001 19 44025 lymph node SUP0081 37997 mix SUP014 3 46740 mixed SUP015 8 46850 mixed DGD001 264 91971lymphocyte DGD004 25 91423 lymphocytes STM001 292 181899 bone marrowOBE01 48 132217 adipocytes

SEQ ID NO: 629 was further analyzed for their presence in the publicdbEST database and their tissue source. SEQ ID NO: 629 was found to beexpressed in following tissues: NIH_MGC_(—)119, NIH_MGC_(—)7,NCI_CGAP_GC6, Soares_testis, NCI_CGAP_Brn25, and NCI_CGAP_Brn35. Furtherdescription of the tissue source can be found athttp://image.llnl.gov/image/html/humlib_info.shtml.

SEQ ID NO: 629 was mapped to human chromosome 1p34.1-35.3 by BLASTanalysis with human genome sequences.

Example 26 Three-Dimensional Structure of 407

The GeneAtlas software package (Molecular Simulations Inc. (MSI), SanDiego, Calif.) was used to predict the three-dimensional structuremodels of SEQ ID NO: 407. Models were generated by (1) PSI-BLAST whichis the multiple alignment sequence profile-based searching developed byAltschul et al, (Nucl. Acids. Res. 25:3389-3408 (1997), (2) HighThroughput Modeling (MSI) which is an automated sequence and structuresearching procedure (Accelrys, Burlington, Mass.), and (3) SeqFold whichis a fold recognition method described by Fischer and Eisenberg (J. Mol.Biol. 209:779-791 (1998)). This analysis was carried out, in part, bycomparing the Serpin-like amino acid sequence (SEQ ID NO: 407) with theknown NMR (nuclear magnetic resonance) and x-ray crystalthree-dimensional structures of stem cell factors as templates. The beststructural model predictions for Serpin-like polypeptides, SEQ ID NO:407, were each based on the stem cell factor templates and the resultsare summarized below, where “PDB ID”, the Protein DataBase (PDB)identifier given to template structure; “Chain ID”, identifier of thesubcomponent of the PDB template structure; “Compound Information”,information of the PDB template structure and/or its subcomponents; “PDBFunction Annotation” gives function of the PDB template as annotated bythe PDB files (Berman et al., Nucl. Acids Res. 28:235-242 (2000), hereinincorporated by reference); start and end amino acid position of theprotein sequence aligned; PSI-BLAST score, the verify score, the SeqFoldscore, and the Potential(s) of Mean Force (PMF) score. The verify scoreis produced by GeneAtlas™ software (MSI), is based on Dr. Eisenberg'sProfile-3D threading program developed in Dr. David Eisenberg'slaboratory (U.S. Pat. No. 5,436,850 and Luthy, Bowie, and Eisenberg,Nature, 356:83-85 (1992)) and a publication by R. Sanchez and A. Sali,Proc. Natl. Acad. Sci. USA, 95:13597-12502. The verify score produced byGeneAtlas normalizes the verify score for proteins with differentlengths so that a unified cutoff can be used to select good models asfollows:Verify score (normalized)=(raw score−½ high score)/(½ high score)

The PFM score, produced by GeneAtlas™ software (MSI), is a compositescoring function that depends in part on the compactness of the model,sequence identity in the alignment used to build the model, pairwise andsurface mean force potentials (MFP). As given in Table 70, a verifyscore between 0 and 1.0, with 1 being the best, represents a good model.Similarly, a PMF score between 0 and 1.0, with 1 being the best,represents a good model. A SeqFold™ score of more than 50 is consideredsignificant. A good model may also be determined by one of skill in theart based all the information in Table 70 taken in totality. TABLE 70SEQ Start End PSI- ID PDB Chain Compound amino amino BLAST Verify PMFSeqFold NO: ID ID Information PDB Function Annotation acid acid scorescore score score 407 1ova Serpin _1ova_ovalbumin_(eggalbumin) 3 4255.1e−61 0.81 1.00

The overall topology of SEQ ID NO: 407 was similar to ov-serpinssubfamily. It exhibited several β-sheets and α-helices. The amino acidsfrom residues 82 through 101 add an insertion of 20 amino acids in oneof the loop regions in the structure of SEQ ID NO: 407. This is likelyimportant to the function of the protein as loop segments are usually onthe surface of proteins, and often provide interfaces forprotein-protein interaction binding sites and enzymatic active sites.

Example 27 Three-Dimensional Structure of SEQ ID NO: 572

The GeneAtlas™ software package (Molecular Simulations, Inc. (MSI), SanDiego, Calif.) was used to predict the three-dimensional structure modelof NGALHy1 polypeptide (SEQ ID NO: 572). Models were generated by (1)PSI-BLAST which is the multiple alignment sequence profile-basedsearching developed by Altschul et al. (Nucl. Acids Res. 25:3389-3408(1997)), (2) High Throughput Modeling (MSI) which is an automatedsequence and structure searching procedure (Accelrys, Burlington,Mass.), and (3) SeqFold which is a fold recognition method described byFischer and Eisenberg (J. Mol. Biol. 209:779-791 (1998)). This analysiswas carried out, in part, by comparing the NGALHy1 amino acid sequence(SEQ ID NO: 572) with the known NMR (nuclear magnetic resonance) andx-ray crystal three-dimensional structure of NGAL (SEQ ID NO: 586) astemplate. The best structural model prediction for NGALHy1 was based onthe NGAL template and the results are summarized below, wherein “PDB ID”is the Protein DataBase (PDB) identifier given to template structure;“Chain ID” is the identifier of the subcomponent of the PDB templatestructure; “Compound Information” is the information of the PDB templatestructure and/or its subcomponents; “PDB Function Annotation” givesfunction of the PDB template as annotated by the PDB files (Berman etal., Nucl. Acids Res. 28:235-242 (2000), herein incorporated byreference); start and end amino acid position of the protein sequencealigned; PSI-BLAST score, the verify score, the SeqFold score, and thePotential(s) of Mean Force (PMF) score. The verify score is produced byGeneAtlas™ software (MSI), is based on Dr. Eisenberg's Profile-3Dthreading program developed in Dr. David Eisenberg's laboratory (U.S.patent Ser. No. 09/5,436,850; Luthy et al., Nature 356:83-85 (1992);Sanchez and Sali, Proc. Natl. Acad. Sci. USA 95:13597-13602 (1998)). Theverify score produced by GeneAtlas™ normalizes the verify score forproteins with different lengths so that a unified cutoff can be used toselect good models as follows:Verify score (normalized)=(raw score− 1/2 high score)/( 1/2 high score)

The PMF score, produced by GeneAtlas™ software (MSI), is a compositescoring function that depends in part on the compactness of the model,sequence identity in the alignment used to build the model, pairwise andsurface mean force potentials (MFP). As given in Table 71 below, averify score between 0 and 1.0, with 1 being the best, represents a goodmodel. Similarly, a PMF score between 0 and 1.0 with 1 being the best,represents a good model. A good model may also be determined by one ofskill in the art based on all the information in Table 71 taken intotality. TABLE 71 Start End PSI SEQ ID PDB Compound PDB Function AminoAmino Blast Verify PMF NO ID Information Annotation Acid Acid Scorescore Score 572 1 NGAL Sugar binding protein. 32 155 3.2e−24 0.4 0.9crystal structure of human neutrophils gelatinase associated lipocalinmonomer. NGAL; neutrophils, NGAL, lipocalin

Example 28 Expression Analysis of SEQ ID NO: 240

First strand human cDNA libraries from multiple tissues were screenedwith gene specific primers for SEQ ID NO: 240(5′-CGATGCAGGAGAACCAGGAC-3′ and 5′-CCTCAGGACCAGTGGGACC-3′). Thecommercial panels (Clontech) screened were: Panel I (heart, brain,placenta, lung, liver, skeletal muscle, kidney and pancreas), Panel II(Spleen, thymus, prostate, testis, ovary, small intestine, colon andadipocyte from a marathon ready cDNA library), immune panel (spleen,lymph node, thymus, tonsil, bone marrow, fetal liver, peripheral bloodleukocyte) and a blood fraction panel (mononuclear, resting CD8+,resting CD4+, resting CD14+, resting CD19+, activated mononuclear cells,activated CD4+ and activated CD8+). PCR was performed for a total of 30cycles using the following conditions: an initial denaturation at 94° C.for 3 min, followed by 5 cycles of 30 s at 94° C., 30 sec at 68° C. and1 min at 72° C., followed by 5 cycles of 30 s at 94° C., 30 sec at 64°C. and 1 min at 72° C., followed by 20 cycles of 30 s at 94° C., 30 secat 60° C. and 1 min at 72° C. followed by an extension of 10 min at 72°C. The amplification product was detected by analysis on agarose gelsstained with ethidium bromide. The SEQ ID NO: 240 was expressed in ahuman adipose tissue cDNA library.

Example 29 Cellular Localization of SEQ ID NO: 241

SEQ ID NO: 240 specific primers corresponding to the translational startregion and the carboxy-terminal region, excluding the stop codon of theSEQ ID NO: 240 sequence, were used (5′-TATAAGCTTATGAGGATCTGGTGGCTTCTG-3′and 5′-AATCTCAGACGGGCTGCTGAACAGAAGG-3′). PCR amplification of the 883 ntproduct was performed using the following conditions; an initialdenaturation at 94° C. for 3 min, followed by 5 cycles of 30 s at 94°C., 30 sec at 66° C. and 1 min at 72° C., followed by 5 cycles of 30 sat 94° C., 30 sec at 62° C. and 1 min at 72° C., followed by 20 cyclesof 30 s at 94° C., 30 sec at 58° C. and 1 min at 72° C. followed by anextension of 10 min at 72° C. These primers generated a fragment of DNAcorresponding to the entire coding region of the SEQ ID NO: 240, flankedby HINDIII and XHOI sites. The PCR product was digested accordingly togenerate overhang ends that were ligated to the HINDIII and XHOI sitesof PCDNA3.1/myc-His(+)A (Invitrogen). The resultant mammalian expressionplasmid (AQL1/myc-His) allows for expression of the AQL1 coding sequencefused in-frame with the myc-6His epitope at the carboxy terminus.

The mammalian expression vector was transfected into COS-7 cells.Briefly, cells in a 10 cm dish with 8 ml of medium were incubated with16 μl of Fugene-6 and 4 μg of DNA for 12 h. The medium was then replacedwith serum-free DMEM and incubated for an additional 48 h prior toharvesting. After the conditioned medium was collected from transfectedCOS-7 cells, cells were washed twice with PBS and then scrapped fromplates. Upon centrifugation, the cells were resuspended in PBScontaining 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin, and 0.2 μg/mlaprotinin. After a brief sonication, the cytosolic fraction wasseparated from the insoluble membrane fraction by centrifugation.Purification of proteins from the cytosolic and from the media tookplace at 4 C in the presence of 100 μl of Ni-NTA resin (Qiagen). Theresin was washed twice with 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, and 5mM imidazole.

To determine the cellular localization of the AQL1/myc-His taggedprotein, Western blot analysis was performed on cytosolic, membrane, andmedium fractions using an anti-myc antibody. AQL1/myc-His tagged proteinwas detected primarily in the medium (85%), but some protein was alsodetected in the cytosolic (10%) and membrane (5%) fractions. Thepredicted molecular mass of the tagged AQL1/myc-His tagged protein is 38kDa. However, the approximate 44 kDa electrophoretic mobility suggeststhat AQL1/myc-His tagged protein is post-translationaly modified.

Example 30 A. Expression of Full-Length Polypeptides of the Invention inCells

Chinese Hamster Ovary (CHO) cells or other suitable cell types are grownin DMEM (ATCC) and 10% fetal bovine serum (FBS) (Gibco) to 70%confluence. Prior to transfection, the media is changed to DMEM and 0.5%FBS. Cells are transfected with cDNAs for SEQ ID NO: 5, 7-13, 15, 17-24,28, 30-156, 160, 162-182, 186, 188-213, 215, 217-239, 241, 243-270, 272,274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355, 357-376, 378,380-401, 408, 410-414, 415, 420, 422-439, 444-480, 482-484, 487,489-501, 505, 507-512, 516, 518-524, 528, 530-539, 542, 544-546, 548,550-553, 557, 559-567, 572, 574, 576, 579, 581-584, 588, 590, 596, 602,604-605, 607, 609-610, 612, 614-615, 618, 620, 622, 624, 626, 628, 630,632, or 634-653 or with pBGal vector by the FuGENE-6 transfectionreagent (Boehringer). In summary, 4 μl of FuGENE-6 is diluted in 100 μlof DMEM and incubated for 5 min. Then, this is added to 1 μl of DNA andincubated for 15 min before adding it to a 35 mm dish of CHO cells. TheCHO cells are incubated at 37° C. with 5% CO₂. After 24 h, media andcell lysates are collected, centrifuged and dialyzed against assaybuffer (15 mM Tris pH 7.6, 134 mM NaCl, 5 mM glucose, 3 mM CaCl₂ andMgCl₂).

B. Expression Study Using Polynucleotides of the Invention

The expression of SEQ ID NO: 1-4, 6, 14, 16, 25-27, 29, 157-159, 161,183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303, 322, 324,345-347, 349, 353-354, 356, 377, 379, 405-407, 409, 418-419, 421,441-443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527, 529, 547,549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606, 608,611, 613, 617, 619, 621, 623, 625, 627, 629, or 631 in various tissuesis analyzed using a semi-quantitative polymerase chain reaction-basedtechnique. Human cDNA libraries are used as sources of expressed genesfrom tissues of interest (adult bladder, adult brain, adult heart, adultkidney, adult lymph node, adult liver, adult lung, adult ovary, adultplacenta, adult rectum, adult spleen, adult testis, bone marrow, thymus,thyroid gland, fetal kidney, fetal liver, fetal liver-spleen, fetalskin, fetal brain, fetal leukocyte and macrophage). Gene-specificprimers are used to amplify portions of SEQ ID NO: 1-4, 6, 14, 16,25-27, 29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273,300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407,409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506, 514-515, 517,526-527, 529, 547, 549, 556, 558, 570-571, 573, 577-578, 580, 587, 589,601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631sequence from the samples. Amplified products are separated on anagarose gel, transferred and chemically linked to a nylon filter. Thefilter is then hybridized with a radioactively labeled (³³P-dCTP)double-stranded probe generated from SEQ ID NO: 1-4, 6, 14, 16, 25-27,29, 157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301,303, 322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409,418-419,421,441-443, 485-486,488,503,504,506,514-515, 517, 526-527, 529,547, 549, 556, 558, 570-571, 573, 577-578, 580, 587, 589, 601, 603, 606,608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631 using a Klenowpolymerase, random-prime method. The filters are washed (highstringency) and used to expose a phosphorimaging screen for severalhours. Bands indicate the presence of cDNA including SEQ ID NO:1-4,6,14,16,25-27,29,157-159,161,183-185, 187, 214, 216, 240, 242, 271,273, 300-301, 303, 322, 324, 345-347, 349, 353-354, 356, 377, 379,405-407, 409, 418-419, 421, 441-443, 485-486, 488, 503, 504, 506,514-515, 517, 526-527, 529, 547, 549, 556, 558, 570-571,573,577-578,580, 587, 589, 601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625,627, 629, or 631 sequences in a specific library, and thus mRNAexpression in the corresponding cell type or tissue.

Example 31 Expression of Full-Length Polypeptides of the Invention in E.coli

SEQ ID NO: 5, 15, 28, 160, 186, 215, 241, 272, 302, 323, 348, 355, 378,408, 420, 444, 487, 505, 516, 528, 542, 548, 557, 572, 579, 588, 602,607, 612, 618, 622, 626, or 630 is expressed in E. Coli by subcloningthe entire coding region into a prokaryotic expression vector. Theexpression vector (pQE16) used is from the QIAexpression® prokaryoticprotein expression system (QIAGEN). The features of this vector thatmake it useful for protein expression include: an efficient promoter(phage T5) to drive transcription, expression control provided by thelac operator system, which can be induced by addition of IPTG(isopropyl-β-D-thiogalactopyranoside), and an encoded histidine, His6,tag comprising a stretch of 6 histidine amino acid residues which canbind very tightly to a nickel atom. The vector can be used to express arecombinant protein with a His6 tag fused to its carboxyl terminus,allowing rapid and efficient purification using Ni-coupled affinitycolumns.

PCR is used to amplify the coding region which is then ligated intodigested pQE16 vector. The ligation product is transformed byelectroporation into electrocompetent E. coli cells (strain M15 [pREP4]from QIAGEN), and the transformed cells are plated onampicillin-containing plates. Colonies are screened for the correctinsert in the proper orientation using a PCR reaction employing agene-specific primer and a vector-specific primer. Positives are thensequenced to ensure correct orientation and sequence. To express thepolypeptide of the invention, a colony containing a correct recombinantclone is inoculated into L-Broth containing 100 μg/ml of ampicillin, 25μg/ml of kanamycin, and the culture is allowed to grow overnight at 37°C. The saturated culture is then diluted 20-fold in the same medium andallowed to grow to an optical density at 600 nm of 0.5. At this point,IPTG is added to a final concentration of 1 mM to induce proteinexpression. The culture is allowed to grow for 5 more hours, and thenthe cells are harvested by centrifugation at 3000×g for 15 minutes.

The resultant pellet is lysed using a mild, nonionic detergent in 20 mMTris HCl (pH 7.5) (B-PER™ Reagent from Pierce), or by sonication untilthe turbid cell suspension turned translucent. The lysate obtained isfurther purified using a nickel-containing column (Ni-NTA spin columnfrom QIAGEN) under non-denaturing conditions. Briefly, the lysate isbrought up to 300 mM NaCl and 10 mM imidazole and centrifuged at 700×gthrough the spin column to allow the His-tagged recombinant protein tobind to the nickel column. The column is then washed twice with WashBuffer (50 mM NaH₂PO₄, pH 8.0; 300 mM NaCl; 20 mM imidazole) and iseluted with Elution Buffer (50 mM NaH₂PO₄, pH 8.0; 300 mM NaCl; 250 mMimidazole). All the above procedures are performed at 4° C. The presenceof a purified protein of the predicted size is confirmed with SDS-PAGE.

Example 32 Expression and Purification of Polypeptides of the Inventionfrom Insect Cells

Polypeptides of the invention are expressed in insect cells as follows:

An open reading frame expressing a polypeptide of the invention iscloned by PCR into a pIB/V5-His TOPO TA cloning vector (InvitrogenCorporation) either with a Myc/His tag or without any tags. Insect cells(High Five TM, Invitrogen) are transfected with the plasmid DNAcontaining the tagged or untagged version of the polypeptide of theinvention by using the InsectSelect™ System (Invitrogen). The expressionof the polypeptide of the invention is determined by transientexpression. The medium containing an expressed polypeptide of theinvention is separated on SDS-PAGE and the expressed polypeptide of theinvention is identified by Western blot analysis. For large-scaleproduction of a polypeptide of the invention, resistant cells areexpanded into flasks containing Ultimate InsectSerum-Free medium(Invitrogen). The cells are shaken at ˜100 mph at 27° C. for 4 days. Theconditioned media containing the protein for purification are collectedby centrifugation.

Example 33 Production of Antibodies Specific to the Polypeptides of theInvention

Cells expressing a polypeptide of the invention are identified usingantibodies specific to the polypeptide of the invention. Polyclonalantibodies are produced by DNA vaccination or by injection of peptideantigens into rabbits or other hosts. An animal, such as a rabbit, isimmunized with a peptide from the extracellular region of thepolypeptide of the invention conjugated to a carrier protein, such asBSA (bovine serum albumin) or KLH (keyhole limpet hemocyanin). Therabbit is initially immunized with conjugated peptide in completeFreund's adjuvant, followed by a booster shot every two weeks withinjections of conjugated peptide in incomplete Freund's adjuvant.Antibodies of the invention are affinity purified from rabbit serumusing a peptide of the invention coupled to Affi-Gel 10 (BioRad), andstored in phosphate-buffered saline (PBS) with 0.1% sodium azide. Todetermine that the polyclonal antibodies are specific for thepolypeptide of the invention, an expression vector encoding thepolypeptide of the invention is introduced into mammalian cells. Westernblot analysis of protein extracts of non-transfected cells and the cellsexpressing the polypeptide of the invention is performed using thepolyclonal antibody sample as the primary antibody and a horseradishperoxidase-labeled anti-rabbit antibody as the secondary antibody.Detection of a band corresponding to the molecular weight of thepolypeptide of the invention in the cells expressing the polypeptide ofthe invention and lack thereof in the control cells indicates that thepolyclonal antibodies are specific for said polypeptide of theinvention.

Monoclonal antibodies are produced by injecting mice with a peptide ofthe invention, with or without adjuvant. Subsequently, the mouse isboosted every 2 weeks until an appropriate immune response has beenidentified (typically 1-6 months), at which point the spleen is removed.The spleen is minced to release splenocytes, which are fused (in thepresence of polyethylene glycol) with murine myeloma cells. Theresulting cells (hybridomas) are grown in culture and selected forantibody production by clonal selection. The antibodies are secretedinto the culture supernatant, facilitating the screening process, suchas screening by an enzyme-linked immunosorbent assay (ELISA).Alternatively, humanized monoclonal antibodies are produced either byengineering a chimeric murine/human monoclonal antibody in which themurine-specific antibody regions are replaced by the human counterpartsand produced in mammalian cells, or by using transgenic “knock out” micein which the native antibody genes have been replaced by human antibodygenes and immunizing the transgenic mice as described above.

Example 33 Multiplex Analysis of Protein Phosphorylation andCytokine/Chemokine Activation After Treatment with Polypeptides of theInvention A. Secretion Levels of the Polypeptide of the Invention

The full-length open reading frame of the polypeptide of the invention(i.e. SEQ ID NO: 2-4, 6, 14, 16, 26-27, 29, 158-159, 161, 184-185, 187,214, 216, 240, 242, 271, 273, 301, 303, 322, 324, 346-347, 349, 354,356, 377, 379, 407, 409, 419, 421, 443, 486, 488, 504, 506, 515, 517,527, 529, 541, 543, 547, 549, 556, 558, 571, 573, 578, 580, 587, 589,601, 603, 606, 608, 611, 613, 617, 619, 621, 623, 625, 627, 630, or 631)is cloned into the mammalian expression vector pcDNA3.1/V5-His-Topo(Invitrogen, Carlsbad, Calif.) to generate a C-terminal V5-His taggedexpression construct. The resulting plasmid is transiently transfectedinto COS7L cells using the Fugene-6 transfection reagent (RocheBiosciences). The presence of the V5-His tagged protein is determined inboth culture supernatant and cell lysate by Western blotting usinganti-V5 antibodies and chemiluminescence visualization. The percentsecretion is determined by comparing the amount of protein in thesupernatant to the amount of protein in the cell lysate.

B. Detection of Intracellular Protein Phosphorylation

The assay described below, a Bio-Plex (Bio-Rad, Hercules, Calif.)phosphorylation assay, is one of several methods employed for measuringprotein phosphorylation in order to assess potential functions ofsecreted proteins in the particular cell type tested. Briefly, purifiedantibodies against various protein kinases, JNK, p38MAPK, erk, Stat3,and IκBα, are conjugated to microsphere sets according to themanufacturer's protocol. Culture supernatant from COS7L cells,transiently transfected with an expression plasmid containing a V5-Histagged fusion protein of the polypeptide of the invention (see Example33A), is harvested and 10 μl of the culture supernatant is added to apanel of target cell lines for 15 min at 37° C. Cells are lysed and thelysate is clarified. The conjugated microspheres are incubated with 25μl of cell lysate in a final volume of 50 μl in a 96-well plateovernight at room temperature with constant shaking. After incubation,the microspheres are washed with Tris buffered saline (TBS) containing0.02% Tween-20 (TBST). Protein phosphorylation is detected by incubatingthe microspheres with 25 μl of a mixture of biotinylated antibodiesagainst the phosphorylated forms of the protein kinases, for example,anti-phospho-Stat3, in TBST containing 5% mouse serum at roomtemperature for 30 min with constant shaking. The microspheres arewashed with TBST and further incubated with 2 μg/ml ofstreptavidin-phycoerythrin (PE). The resulting microspheres with thereaction complex are analyzed using the Luminex Reader (Luminex Co.,Austin, Tex.).

C. Detection of Cytokine/Chemokine Levels

Cytokine and chemokine levels are determined using the assay describedbelow, the Luminex Multi-plex bead assay, which is very similar to atypical sandwich ELISA assay, but utilizes Luminex microspheresconjugated to anti-cytokine and anti-chemokine antibodies (Vignali, J.Immunol. Methods 243:243-255 (2000), herein incorporated by reference).Briefly, purified antibodies against a variety of cytokines andchemokines are conjugated to microsphere sets (Luminex Co., Austin,Tex.) according to the manufacturer's protocol. Culture supernatant fromCOS7L cells, transiently transfected with an expression plasmidcontaining a V5-His tagged fusion protein of the invention (see Example33A), is harvested and 25 μl of the culture supernatant is added to apanel of target cell lines and incubated overnight at 37° C. Conditionmedia is then harvested. The conjugated microspheres are incubated with50 μl in a 96-well filter plate at room temperature for 30 min withconstant shaking. After incubation, the microspheres are washed andincubated with 50 μl (1 μg/ml) of biotinylated anti-cytokine oranti-chemokine antibodies in phosphate buffered saline (PBS) containing0.5% Tween-20, 0.2% BSA, 5% mouse serum at room temperature for 30 min.The microspheres are washed and further incubated with 2 μg/ml ofStreptavidin-PE. The resulting microspheres with the reaction complexare analyzed using the Luminex Reader (Luminex Co., Austin, Tex.).

Example 34 Calcium Mobilization Assay

Many extracellular signals to intracellular targets are mediated byincreases in free calcium levels in the cytoplasm. Calcium mobilizationfrom intracellular stores can be detected in many cell types by loadingthe cells with a Ca²⁺ sensitive indicator such as fura-2-AM. Theincrease in fluorescence is detected by a fluorescence plate reader.Cells will be incubated in media containing 5 μM Fura-2 AM, 5 μMPluronic F-127 for 30 min. After the addition of adiponectin-likeprotein the Fura-2 intensity will be monitored approximately every 20sec by a fluorescent plate reader (Molecular Dynamics) and compared tothe intensity of cells with basal calcium levels.

Example 35 Fatty Acid Oxidation Assay

The oxidation of palmitate or oleate in culture C2Cl2 skeletal musclecells (ATCC; CRL-1772) upon exposure to AQL1 protein is measuredaccording to published procedures (Barger et al., J. Clin. Invest.105:1723-1730 (2000)). In summary, nearly confluent C2C12 myocytes arekept in differentiation medium (DMEM, 2.5% horse serum) for 7 days, atwhich time formation of myotubes is maximal. [1-¹⁴C]oleic acid (1μCi/ml) is added to the cells and incubated for 90 minutes at 37° C. inthe absence/presence of adiponectin-like protein. In some of the assaysa proteolytically cleaved adiponectin-like protein (cleaved betweenlysine 190-glycine 191) may be employed. During the experiment the C2C12cells are incubated in a closed system containing Whatman paper tocollect the ¹⁴CO₂ gas released during fatty acid oxidation. After theincubation the Whatman paper is removed and the amount of ¹⁴Cradioactivity is determined by liquid scintillation counting.

Example 36 Macrophage Phagocytosis Assay

Human macrophages are incubated in the presence/absence ofadiponectin-like protein for 24 hours at 37° C. in 96-well plates.Fluobrite fluorescent-microspheres (0.75 G; Polyscience, Warrington,Pa.) are added to each well, followed by one hour incubation at 37° C.Nonadherent latex beads are removed by gentle washing and the cells areincubated for an additional 30 minutes to complete phagocytosis. Thecells are harvested by short-time treatment with EDTA and trypsin andwashed vigorously three times with PBS to remove noningested beads. Theamount of ingested beads will be measured with a FACScan.

Example 37 Glucose Uptake Assay

The adiponectin-like proteins influence carbohydrate and lipidmetabolism. One of the ways by which the adiponectin-like proteinsaffect the development of insulin resistence is by altering glucosemetabolism. To evaluate the effect of the polypeptides of the inventionon glucose uptake. differentiated rat L6 myotube cells are cultured in96-well plate for a minimum of 5 days in DMEM with 3% horse serum. Thecells are incubated in 100 μl serum free media containing 25 mM glucoseat 37 C in 5% CO₂ with or without adiponectin-homolog proteins of SEQ IDNO: 2 or 8 at a concentration of 30 μg/ml for 4-5 hours, followed by asubsequent incubation with insulin (100 nM) for 1 hour. The cells arethen washed with serum containing media twice to remove glucose. Thecells are further incubated with 10 μM [1,2-³H]2-deoxyglucose in 50 μlHBS for 20 min at 30 C. The overlayed media is removed and the cells arewashed twice with 2001 μl of HBS buffer to remove the excess2-Deoxy-D-[1-³H]2-glucose from the cells. The cells are lysed with 100μl of 1 M NaOH by incubation for 30 min. The supernatants from the cellsare collected and stored. 5 μl of supernatant is transferred to a96-well plate for radioactive counting in the 96-well scintillationcounter for measuring the ³H uptake by the cells. The ³H uptake by cellsreflects the glucose uptake induced by adiponectin by the cells.(Sarabia et. al., Biochem Cell Biol 68:536-542 (1990); Yu et al., J.Biol. Chem. 276: 19994-19998 (2001)).

Example 38 Effects on Neuronal Growth In Vitro and In Vivo A. FibroblastSpreading Assays

Mouse NIH 3T3 cells are cultured and assayed for spreading behavior inDMEM containing 10% FCS, usually to a maximum of 70-80% confluency.Subconfluent 3T3 cells are plated for 1 h in serum-containing mediabefore fixation and staining with rhodamine-phalloidin. Glass coverslipsare precoated with poly-L lysine, washed, and coated with PBS containingNogo peptides, the soluble ectodomain of NgR, the soluble ectodomain ofNgRHy, or anti-NgRHy antibodies, the soluble domain of neural IgCAM-likepolypepides (i.e. SEQ ID NO: 501, 512, or 539), neural IgCAM-likepeptides (i.e. SEQ ID NO: 490, 508, 519, or 531), or anti-neuralIgCAM-like antibodies. Appropriate concentration of protein for coatingwill be predetermined in separate assays. The protein drops are allowedto dry, the slides washed in PBS, and then fixed in 1% glutaraldehyde inPBS or 4% paraformaldehyde in PBS.

B. Growth Cone Collapse Assays

For the assessment of growth cone collapse, chick DRGs from embryonicday 7 (E7) are explanted onto laminin-coated chamber slides in F12medium with 10 ng/ml nerve growth factor and 10% fetal bovine serum for20 h. Phosphate-buffered saline (PBS) solutions containing 1 mM DTT withor without the soluble ectodomain of NgRHy, NgRm or neural IgCAM-likepolypeptides are added to the explants (225 μl) and incubated at 37° C.for one hour. For each explant, all growth cones were scored ascollapsed or fan-shaped (Igarashi et al., Science 259: 77.1993). ForE7-E15 cultures, the origin of neuronal cells can be assessed bystaining with anti-neurofilament antibodies and the O4 antibody fordetection of oligodendrocytes. Neurites are traced by observation ofrhodamine-phalloidin staining of F-actin in processes.

In the above assays, neurite outgrowth or inhibition of growth conecollapse may increase after the addition of the NgRHy or neuralIgCAM-like polypeptide as compared to cultures lacking added peptide orcultured in the presence of Nogo protein. This indicates that NgRHy orneural IgCAM-like peptide acts as an antagonist to the endogenous NgRprotein and inhibits the effects of Nogo proteins on preventing neuralgrowth.

C. Neuronal Co-Culture Assays

Embryonic DRG neurons are co-cultured with adult oligodendrocytes(Oudega et al., Neuroscience 100: 873-883. 2000) in the presence of thesoluble ectodomain of NgRHy or neural IgCAM-like polypeptides orantibodies specific for NgRHy or neural IgCAM-like polypeptides todetermine the antagonistic effects of NgRHy or neural IgCAM-likepeptides and antibodies.

Adult oligodendrocytes are isolated from rat spinal cord and cultured inDMEM/F12 with 0.5 μg NGF, 15 nM selenium, 1 mg transferrin, 0.5 μginsulin-like growth factor-1 and 2.5% heat-inactivated bovine serum incollagen coated Aclar hats (2×10⁴ cell/hat). After 4 days theoligodendrocytes are incubated in either plain DMEM or DMEM containingNgRHy or neural IgCAM-like polypeptides (at a pre-determined optimalconcentration) for 30 min at 37° C. Following this incubation, 75% ofthe media is replaced by a suspension of DRG neurons (10⁴ cells/hat)isolated as described above. The co-cultures are maintained inneurobasal medium [B27 suppl. (Gibco BRL), containing 50 ng/ml partiallypurified NGF and 50 ug/ml ascorbic acid] at 37° C. 5% CO₂ for 72 h. Thecultures are rinsed in L15 media with 10% normal goat serum and stainedwith mouse O1 anti-oligodendrocyte antibody. The oligodendrocytes arevisualized using a rhodamine-conjugated goat-anti-mouse antibody. Thecells are then fixed in 4% paraformaldehyde in PBS and permeabilizedwith 0.2% Triton X-100. To visualize axons, the co-culture is stainedwith an anti-neurofilament antibody.

The effects of NgRHy or neural IgCAM-like peptides and antibodies on DRGneurite growth can be quantified in a 2.0×0.5 mm strip by measuring thelength of neurites that are touched an oligodendrocyte. An increase inneurite outgrowth in the co-culture in the presence of NgRHy or neuralIgCAM-like peptides or antibodies demonstrates that NgRHy or neuralIgCAM-like peptides or antibodies can act as antagonists to theendogenous receptor and prevent the inhibition of neural growth mediatedby the oligodendrocytes, indicating NgRHy or neural IgCAM-like peptidesand antibodies can be an effective treatment in vivo for the promotionof neural regeneration.

D. Neurite Outgrowth Assays

For the assessment of neurite outgrowth, chick DRGs from embryonic day 7(E7) are explanted onto laminin-coated chamber slides in F12 medium with10 ng/ml nerve growth factor and 10% fetal bovine serum for 20 h.Phosphate-buffered saline (PBS) solutions containing 1 mM DTT with orwithout the soluble domain of neural IgCAM-like polypeptides are addedto the explants (225 μl) and incubated at 37° C. for one hour. For eachexplant, all neurite are scored as collapsed or fan-shaped (Igarashi etal., Science 259: 77. 1993). For E7-E15 cultures, the origin of neuronalcells can be assessed by staining with anti-neurofilament antibodies andthe O4 antibody for detection of oligodendrocytes. Neurites are tracedby observation of rhodamine-phalloidin staining of F-actin in processes.

In the above assays, neurite outgrowth may increase after the additionof the neural IgCAM-like polypeptide as compared to cultures lackingadded peptide indicating that neural IgCAM-like peptides enhance neuriteoutgrowth.

Example 39 Assessment of Binding Partners for NgRHy

The NgRHy polynucleotide sequence can be transfected into host cells asdescribed previously. For instance, NgRHy is transfected into either COScells or 3T3 fibroblasts and the binding of NgRHy to the Nogo proteinand its isoforms are assessed by means well-known in the art. Forexample, the Nogo protein is labeled with a detectable label such asalkaline phosphatase (Fournier et al., supra) or conjugated to biotinmolecules or a fluorophore such as fluoroisothiocyanate (FITC) orphycoerythrin (PE). The binding of labeled Nogo protein to cellsexpressing NgRHy is assessed by an appropriate detection method based onthe labeled Nogo protein. These methods include staining for thepresence of Nogo on cells bound to a coverslip or through flowcytometric analysis of the fluorophore-conjugated Nogo protein to theNgRHy-expressing cells.

The NgRHy of the invention can also be expressed in neurons such asembryonic DRG and retinal neurons which demonstrate a weak Nogo-66response, in order to assess the ability of NgRHy to mediate Nogoactivity (Fournier et al., supra). Infection of neurons with HSVcontaining NgRHy is carried out according to Takahashi et al, NatureNeurosci. 1: 487-493. 1998. cDNA of the invention can be inserted intoan plasmid such as pHSV-PrpUC containing the immediate early promoter ofHSV and an HSV packaging site. The plasmid is transfected into the HSVpackaging cell line 2-2 which is infected after 24 hrs with areplication deficient HSV, such as the IE2 deletion mutant 5dl1.2.Recombinant viral stocks are amplified by sequential rounds ofinfection. Viral stocks are added to neuronal cultures at aconcentration of approximately 106 plaque forming units (PFU)/ml 24hours before analysis of neuronal culture. Neuronal growth is assayed asdescribed previously.

Example 40 Effect of Neural IgCAM-Like Polypeptides on Astrocyte CellProliferation

Primary astrocytes are trypsinized and transferred to 96-well plates ata density of 2×10⁵ cells/ml. After cell attachment for 24 h, the culturemedium is exchanged for serum-free medium. After 48 h, neural IgCAM-likepolypeptides are added. After 12 h, [³H]thymidine is added (10 μCi/ml)and the incubation proceeds for another 12 h. Incorporation of[³H]thymidine is measured and cells are harvested onto glass filtersusing an automatic cell harvester (Packard, Meriden, Conn.). Theincorporated radioactivity is measured using a liquid scintillationcounter.

Example 41 Radioimmunoassay for NGAL-Like Activity

To measure the serum levels of NGAL-like polypeptides in normal anddisease states, a radioimmunoassay specific for NGAL-like polypeptidesis used (see Xu et al., J. Immunol. Meth. 171:245-252 (1994), hereinincorporated by reference). Briefly, blood is drawn from patients andgranules are prepared from buffy coats of granulocytes. An equal volumeof 2% Dextran T-500 in phosphate buffered saline (PBS) without magnesiumor calcium is added to the buffy coats for 1 h at room temperature. Thegranulocytes are sedimented and spun through 0.34 M sucrose. The cellsare homogenized in a Potter-Elvehjem homogenizer and mixed with an equalvolume of 0.34 M sucrose and 0.3 M NaCl, clarified at 450×g and thensedimented at 10,000×g. Granulocytes are extracted with 0.05 M aceticacid, pH 4.5 for 1 h at 4° C. and then an equal volume of 0.4 M sodiumacetate, pH 4.0 is added and incubated for 3 h at 4° C., after which thegranules are released into the supernatant (spin at 12,000×g) andconcentrated using an Amicon YM-10 membrane.

NGAL-like polypeptides are radiolabeled with ¹²⁵I according to thechloramines-T method and purified using gel filtration (Hunter et al.,Nature 194:495 (1962), herein incorporated by reference). 50 μl of thesample or standard is sequentially mixed with 50 μl of [¹²⁵I]-NGALHy1 or[¹²⁵ ]-NGALHy2 (8 μl/L), 50 μl anti-NGAL-like antibody [diluted 1:3800in assay buffer (0.05 M sodium phosphate, pH 7.4 containing 0.08 M NaCl,0.01 M Na-EDTA, 0.2% BSA, 0.02% NaN₃, 0.2% CTAB(N-cetyl-N,N,N-trimethylammonium bromide), 0.5% Tween-20)] and incubatedfor 3 h at room temperature. The mixture is further incubated with 2 mlanti-rabbit IgG-Sepharose for 30 min at room temperature. TheNGAL-like-antibody complexes bound to separose are pelleted at 4000 rpm,10 min and the amount of [¹²⁵I]-NGALHy1 or [¹²⁵I]-NGALHy2 is counted ina gamma counter.

Example 42 Anti-Microbial Activity of NGAL-Like Polypeptides

A standard halo assay is used to determine the inhibitory effect ofNGAL-like polypeptides on microbial cell growth by measuring the size ofthe zone of exclusion (see Bjorck et al., Nature 337:385-386 (1989),herein incorporated by reference). This assay can be performed with bothbacterial and yeast strains. For example, Streptococci are plated on0.8% purified agar in Todd-Hewitt broth containing 4% sheep's blood.Sterile filter paper discs are impregnated with either an NGAL-likepolypeptide solution (at various concentrations) or antibiotics, such as0.1 μg benzylpenicillin or 0.2 IU bacitracin, or 1% dimethyl sulfoxide(DMSO) as a control and placed on the plate. The plates are incubated at37° C. for 1-2 days and the sizes of the zones of exclusion (“halos”)are measured, the larger the halo, the greater the inhibition of growth.

Example 43 NGAL-Like Polypeptide Effects on Cell Proliferation A.Lymphoproliferation Assay

To examine the effect of NGAL-like polypeptides on proliferativeresponses, a mitogen-induced lymphoproliferation assay is done (seeCheresh et al., Immunology 51:541-548 (1984), herein incorporated byreference). Mononuclear cells are isolated from blood samples. In amulti-well culture dish, a variety of mitogens are added (one mitogenper well) such as 0.01 mg/ml Concanavalin A (ConA), 0.1 mg/mlphytohaemagglutinin-P, 0.5 mg/ml pokeweed mitogen, or as a control 0.01ml growth medium. To each well is added 1×10⁵ mononuclear cells in 0.19ml growth medium with 10% normal human serum with or without NGAL-likepolypeptides and incubated for 72 h at 37° C. with 5% CO₂. 18 h beforeharvesting, the cells are pulsed with [³H]-thymidine (50 μCi/ml). Cellsare harvested and collected on glass fiber filter paper and lysed withdeionized water. Incorporated thymidine is measured in a scintillationcounter. Data is presented as counts per minute of experimental cultureswith mitogen minus counts per minute of control cultures withoutmitogen.

B. Redistribution of Cell Surface Receptors

Another proliferative response is the redistribution of cell surfacereceptors such as the ConA receptor and cell surface immunoglobulin(sIg) molecules. This effect is measured with a capping assay (seeCheresh et al., Immunology 51:541-548 (1984), herein incorporated byreference). Mononuclear cells (2×10⁶) are incubated for 1 h at 37° C.with 0.2 ml normal human serum with or without NGAL-like polypeptides.Cells are washed with Hank's buffered salt solution without magnesium orcalcium (HBSS) and resuspended in 0.2 ml fluorescein (FITC)-conjugatedConA (15 μg/ml) in HBSS- and incubated for various times at 37° C. Cellsare fixed with 4% paraformaldehyde, washed with PBS, pH 7.4 containing 1mg/ml BSA and placed on a microscope slide. The number of caps isdetermined using a Nikon Optiphot microscope equipped withepifluoresence. A minimum of 200 cells is counted and capped cells aredefined to be cells with uniform fluorescence over less than or equal toone-third of the cell membrane. Alternatively, cells are incubated with0.1 ml FITC-conjugated goat-anti-human Ig (100 μg/ml) in Media 199 for 1h at 4° C., washed with Media 199 and incubated at room temperature for10 min. Cells are fixed in 4% paraformaldehyde, washed with Media 199containing 0.1% gelatin and mounted on microscope slides. Cells arecounted for capping as stated above. Capping is not required forlymphocyte activation, but inhibition of capping may affect other eventsinvolving receptor mobility that may be necessary for triggeringlymphoproliferative responses. Thus, a decrease in capping is associatedwith an inhibition of lymphoid activation as well as cell proliferation.

Example 44 NGAL-Like Polypeptide-Dependent Modulation of MatrixMetalloproteinase Activity

To monitor the effect of NGAL-like polypeptides on matrixmetalloproteinase (MMP) activity, recombinant versions of NGALHy1,NGALHy2, and MMPs, such as MMP-9, are used in an MMP activity assay (seeYan et al., J. Biol. Chem. 276:37258-37265 (2001), herein incorporatedby reference). MMP-9 is diluted in gelatinase buffer (50 mM Tris-HCl, pH7.0, containing 5 mM CaCl₂, 1 μM ZnCl₂) to 0.1 μM and incubated at 37°C. for various times. Aliquots of MMP-9 (10 ng) are collected atdifferent time points and subjected to gelatin zymography (Braunhut andMoses, J. Biol. Chem. 269:13472-13479 (1994), herein incorporated byreference). Briefly, Type 1 gelatin is added to the standard Laemmliacrylamide gel mixture at 1 mg/ml. Samples are mixed 3:1 with thesubstrate gel sample buffer (10% SDS, 4% sucrose, 0.25 M Tris-HCl, pH6.8, and 0.1% bromphenol blue) and loaded onto the gel without boiling.After electrophoresis, gels are soaked in 2.5% Triton X-100 for 30 minand rinsed and incubated overnight at 37° C. in substrate buffer (50 mMTris-HCl, pH 8, 5 mM CaCl₂, and 0.02% NaN₃). Gels are stained in 0.5%Coomassie Blue for 15-30 min and destained in water. MMP activity isvisualized as zones of clearance within the gels and quantitated usingdensitometry.

To analyze NGAL-like-dependent protection, NGALHy1 or NGALHy2 is dilutedin gelatinase buffer and mixed with MMP9 in different molar ratiosranging from 10:1 to 1:20 and incubated at 37° C. for varying times from0.5 to 2 h. Aliquots are collected at each timepoint and degradation ofMMP9 is monitored by substrate electrophoresis. This assay can also beperformed using anti-NGAL-like antibodies.

Investigation of the MMP-NGAL-like interaction is performed in cellculture as well (see Yan et al., J. Biol. Chem. 276:37258-37265 (2001),herein incorporated by reference). Stably transfected cells expressingNGALHy1 or NGALHy2, such as MDA-MB0231 breast carcinoma cells, areincubated with serum-free media at 90% confluency for 20 h. Conditionedmedia is harvested, clarified and electrophoresed to detect MMP9activity. Steady-state mRNA levels of MMP9, tissue inhibitor ofmetalloproteinases-1 (TIMP-1) and a housekeeping geneglyceraldehyde-3-phosphate dehydrogenase (GAPDH) are determined byquantitative real-time PCR analysis (Simpson, et al., Molec. Vision6:178-183 (2000), herein incorporated by reference) to determine therelative copy number of MMP9 mRNA expressed per cell. Thus, a comparisonof MMP9 protein and mRNA levels can be made to determine the effect ofNGAL-like polypeptides on MMP9 activity.

Example 45 Apoptosis Assay

Apoptosis is the controlled process by which cells under a programmedcell death. Apoptosis is studied by analysis of dead or dying cells andone of the methods to to identify dying or apoptotic cells by TUNELassayacridine orange and LysoTracker staining. The TUNEL, acrdine orangeand LysoTracker staining assays are performed as described in Hersh etal (Proc. Natl. Acad. Sci. USA. 99:4355-4360 (2002), incorporated hereinby reference).

Example 46 Endocytosis Assays

Endocytosis is a process by which the cells internalize proteins orlipids from the extracellular space to the cytoplasm. This process ischaracterized by several steps which include binding of the protein orlipid to a receptor, interalization of the bound material and transferto sorting endosomes followed by transfer of the protein or lipid fromsorting endosomes to lysosomes. These steps are studied by a variety ofdifferent assays that are trace the movement of lipids or proteins thatare tagged with a radiolabeled or fluorescent marker. Detection of theradiolabeled or fluorescent marker is done at every step of theendocytosis pathway by fluorescence microscopy or by measuring theradioactivity associated with whole cells or isolated fractions of thecells containing the plasma membrane, endosomes, lysosomes, golgicomplex etc. A combination of the assays is used to study endocytosis asdescribed in Chen et al. (Proc. Natl. Acad. Sci USA, 95:6373-6378(1998), incorporated herein by reference).

Example 47 Expression Levels of Peroxidasin-Like mRNA in Various TumorCell Lines

Expression of peroxidasin-like mRNA is determined in various tumor celllines, including lymphoma, leukemia, melanoma, breast cancer, ovariancancer, lung cancer, brain cancer, etc., and tumor tissues. Poly-Amessenger RNA is isolated from the cell lines and subjected toquantitative, real-time PCR analysis (Simpson, et al., Molec. Vision. 6:178-183 (2000), herein incorporated by reference) to determine therelative copy number of peroxidasin-like mRNA expressed per cell in eachline. Elongation factor 1 mRNA expression is used as a positive controland normalization factors in all samples.

Expression of peroxidasin-like mRNA is determined in various healthy andtumor tissues. Poly-A mRNA is isolated from various tissues andsubjected to quantitative, real-time PCR analysis, as described above,to determine the relative expression of peroxidasin-like mRNA in thesample.

Example 48 In Vitro Antibody-Dependent Cytotoxicity Assay

The ability of a peroxidasin-like protein-specific antibody to induceantibody-dependent cell-mediated cytoxicity (ADCC) is determined invitro. ADCC is performed using the CytoTox 96 Non-Radioactive CytoxicityAssay (Promega; Madison, Wis.) (Hornick et al., Blood 89:4437-4447,(1997)) as well as effector and target cells. Peripheral bloodmononuclear cells (PBMC) or neutrophilic polymorphonuclear leukocytes(PMN) are two examples of effector cells that can be used in this assay.PBMC are isolated from healthy human donors by Ficoll-Paque gradientcentrifugation, and PMN are purified by centrifugation through adiscontinuous percoll gradient (70% and 62%) followed by hypotonic lysisto remove residual erythrocytes. RA1 B cell lymphoma cells (for example)are used as target cells.

RA1 cells are suspended in RPMI 1640 medium supplemented with 2% fetalbovine serum and plated in 96-well V-bottom microtitier plates at 2×10⁴cells/well. peroxidasin-like protein-specific antibody is added intriplicate to individual wells at 1 μg/ml, and effector cells are addedat various effector:target cell ratios (12.5:1 to 50:1). The plates areincubated for 4 hours at 37° C. The supernatants are then harvested,lactate dehydrogenase release determined, and percent specific lysiscalculated using the manufacture's protocols.

Example 49 Toxin-Conjugated Peroxidasin-Like Protein-Specific Antibodies

Antibodies to peroxidasin-like protein are conjugated to toxins and theeffect of such conjugates in animal models of cancer is evaluated.Chemotherapeutic agents, such as calicheamycin and carboplatin, or toxicpeptides, such as ricin toxin, are used in this approach. Antibody-toxinconjugates are used to target cytotoxic agents specifically to cellsbearing the antigen. The antibody-toxin binds to these antigen-bearingcells, becomes internalized by receptor-mediated endocytosis, andsubsequently destroys the targeted cell. In this case, theantibody-toxin conjugate targets peroxidasin-like protein-expressingcells, such as B cell lymphomas, and deliver the cytotoxic agent to thetumor resulting in the death of the tumor cells.

One such example of a toxin that may be conjugated to an antibody iscarboplatin. The mechanism by which this toxin is conjugated toantibodies is described in Ota et al., Asia-Oceania J. Obstet. Gynaecol.19: 449-457 (1993). The cytotoxicity of carboplatin-conjugatedperoxidasin-like protein-specific antibodies is evaluated in vitro, forexample, by incubating peroxidasin-like protein-expressing target cells(such as the RA1 B cell lymphoma cell line) with various concentrationsof conjugated antibody, medium alone, carboplatin alone, or antibodyalone. The antibody-toxin conjugate specifically targets and kills cellsbearing the peroxidasin-like protein antigen, whereas, cells not bearingthe antigen, or cells treated with medium alone, carboplatin alone, orantibody alone, show no cytotoxicity.

The antitumor efficacy of carboplatin-conjugated peroxidasin-likeprotein-specific antibodies is demonstrated in in vivo murine tumormodels. Five to six week old, athymic nude mice are engrafted withtumors subcutaneously or through intravenous injection. Mice are treatedwith the peroxidasin-like protein-carboplatin conjugate or with anon-specific antibody-carboplatin conjugate. Tumor xenografts in themouse bearing the peroxidasin-like protein antigen are targeted andbound to by the peroxidasin-like protein-carboplatin conjugate. Thisresults in tumor cell killing as evidenced by tumor necrosis, tumorshrinkage, and increased survival of the treated mice.

Other toxins are conjugated to peroxidasin-like protein-specificantibodies using methods known in the art. An example of a toxinconjugated antibody in human clinical trials is CMA-676, an antibody tothe CD33 antigen in AML which is conjugated with calicheamicin toxin(Larson, Semin. Hematol. 38(Suppl 6):24-31 (2001)).

Example 50 Radioimmunotherapy Using Peroxidasin-Like Protein-SpecificAntibodies

Animal models are used to assess the effect of antibodies specific toperoxidasin-like protein as vectors in the delivery of radionuclides inradioimmunotherapy to treat lymphoma, hematological malignancies, andsolid tumors. Human tumors are propagated in 5-6 week old athymic nudemice by injecting a carcinoma cell line or tumor cells subcutaneously.Tumor-bearing animals are injected intravenously with radio-labeledanti-peroxidasin-like protein antibody (labeled with 30-40 μCi of ¹³¹I,for example) (Behr, et al., Int. J. Cancer 77: 787-795 (1988)). Tumorsize is measured before injection and on a regular basis (i.e. weekly)after injection and compared to tumors in mice that have not receivedtreatment. Anti-tumor efficacy is calculated by correlating thecalculated mean tumor doses and the extent of induced growthretardation. To check tumor and organ histology, animals are sacrificedby cervical dislocation and autopsied. Organs are fixed in 10% formalin,embedded in paraffin, and thin sectioned. The sections are stained withhematoxylin-eosin.

Example 51 Immunotherapy Using Peroxidasin-Like Protein-SpecificAntibodies

Animal models are used to evaluate the effect of peroxidasin-likeprotein-specific antibodies as targets for antibody-based immunotherapyusing monoclonal antibodies. Human myeloma cells are injected into thetail vein of 5-6 week old nude mice whose natural killer cells have beeneradicated. To evaluate the ability of peroxidasin-like protein-specificantibodies in preventing tumor growth, mice receive an intraperitonealinjection with peroxidasin-like protein-specific antibodies either 1 or15 days after tumor inoculation followed by either a daily dose of 20 μgor 100 μg once or twice a week, respectively (Ozaki, et al., Blood90:3179-3186 (1997)). Levels of human IgG (from the immune reactioncaused by the human tumor cells) are measured in the murine sera byELISA.

The effect of peroxidasin-like protein-specific antibodies on theproliferation of myeloma cells is examined in vitro using a ³H-thymidineincorporation assay (Ozaki et al., supra). Cells are cultured in 96-wellplates at 1×10⁵ cells/ml in 100 μl/well and incubated with variousamounts of peroxidasin-like protein antibody or control IgG (up to 100μg/ml) for 24 h. Cells are incubated with 0.5 μCi ³H-thymidine (NewEngland Nuclear, Boston, Mass.) for 18 h and harvested onto glassfilters using an automatic cell harvester (Packard, Meriden, Conn.). Theincorporated radioactivity is measured using a liquid scintillationcounter.

The cytotoxicity of the peroxidasin-like protein monoclonal antibody isexamined by the effect of complements on myeloma cells using a⁵¹Cr-release assay (Ozaki et al., supra). Myeloma cells are labeled with0.1 mCi ⁵¹Cr-sodium chromate at 37° C. for 1 h. ⁵¹Cr-labeled cells areincubated with various concentrations of peroxidasin-like proteinmonoclonal antibody or control IgG on ice for 30 min. Unbound antibodyis removed by washing with medium. Cells are distributed into 96-wellplates and incubated with serial dilutions of baby rabbit complement at37° C. for 2 h. The supernatants are harvested from each well and theamount of ⁵¹Cr released is measured using a gamma counter. Spontaneousrelease of ⁵¹Cr is measured by incubating cells with medium alone,whereas maximum ⁵¹Cr release is measured by treating cells with 1% NP-40to disrupt the plasma membrane. Percent cytotoxicity is measured bydividing the difference of experimental and spontaneous ⁵¹Cr release bythe difference of maximum and spontaneous ⁵¹Cr release.

Antibody-dependent cell-mediated cytotoxicity (ADCC) for theperoxidasin-like protein monoclonal antibody is measured using astandard 4 h ⁵¹Cr-release assay (Ozaki et al., supra). Splenicmononuclear cells from SCID mice are used as effector cells and culturedwith or without recombinant interleukin-2 (for example) for 6 days. ⁵¹Cr-labeled target myeloma cells (1×10⁴ cells) are placed in 96-wellplates with various concentrations of anti-peroxidasin-like proteinmonoclonal antibody or control IgG. Effector cells are added to thewells at various effector to target ratios (12.5:1 to 50:1). After 4 h,culture supernatants are removed and counted in a gamma counter. Thepercentage of cell lysis is determined as above.

Example 52 Peroxidasin-Like Protein-Specific Antibodies asImmunosuppressants

Animal models are used to assess the effect of peroxidasin-likeprotein-specific antibodies to suppress autoimmune diseases, such asarthritis or other inflammatory conditions, or rejection of organtransplants. Immunosuppression is tested by injecting mice with horsered blood cells (HRBCs) and assaying for the levels of HRBC-specificantibodies (Yang, et al., Int. Immunopharm. 2:389-397 (2002)). Animalsare divided into five groups, three of which are injected with anti-TLR9antibodies for 10 days, and 2 of which receive no treatment. Two of theexperimental groups and one control group are injected with eitherEarle's balanced salt solution (EBSS) containing 5-10×10⁷ HRBCs or EBSSalone. Anti-peroxidasin-like protein antibody treatment is continued forone group while the other groups receive no antibody treatment. After 6days, all animals are bled by retro-orbital puncture, followed bycervical dislocation and spleen removal. Splenocyte suspensions areprepared and the serum is removed by centrifugation for analysis.

Immunosupression is measured by the number of B cells producingHRBC-specific antibodies. The Ig isotype (for example, IgM, IgG1, IgG2,etc.) is determined using the IsoDetect™ Isotyping kit (Stratagene, LaJolla, Calif.). Once the Ig isotype is known, murine antibodies againstHRBCs are measured using an ELISA procedure. 96-well plates are coatedwith HRBCs and incubated with the anti-HRBC antibody-containing seraisolated from the animals. The plates are incubated with alkalinephosphatase-labeled secondary antibodies and color development ismeasured on a microplate reader (SPECTRAmax 250, Molecular Devices) at405 nm using p-nitrophenyl phosphate as a substrate.

Lymphocyte proliferation is measured in response to the T and B cellactivators concanavalin A and lipopolysaccharide, respectively (Jiang,et al., J. Immunol. 154:3138-3146 (1995). Mice are randomly divided into2 groups, 1 receiving anti-peroxidasin-like protein antibody therapy for7 days and 1 as a control. At the end of the treatment, the animals aresacrificed by cervical dislocation, the spleens are removed, andsplenocyte suspensions are prepared as above. For the ex vivo test, thesame number of splenocytes are used, whereas for the in vivo test, theanti-peroxidasin-like protein antibody is added to the medium at thebeginning of the experiment. Cell proliferation is also assayed usingthe ³H-thymidine incorporation assay described above (Ozaki, et al.,Blood 90: 3179 (1997)).

Example 53 Cytokine Secretion in Response to Peroxidasin-Like ProteinPeptide Fragments

Assays are carried out to assess activity of fragments of theperoxidasin-like protein, such as the Ig domain, to stimulate cytokinesecretion and to stimulate immune responses in NK cells, B cells, Tcells, and myeloid cells. Such immune responses can be used to stimulatethe immune system to recognize and/or mediate tumor cell killing orsuppression of growth. Similarly, this immune stimulation can be used totarget bacterial or viral infections. Alternatively, fragments of theperoxidasin-like protein that block activation through theperoxidasin-like protein receptor may be used to block immunestimulation in natural killer (NK), B, T, and myeloid cells.

Fusion proteins containing fragments of the peroxidasin-like protein,such as the Ig domain (peroxidasin-like-Ig), are made by inserting aCD33 leader peptide, followed by a peroxidasin-like protein domain fusedto the Fc region of human IgG1 into a mammalian expression vector, whichis stably transfected into NS-1 cells, for example. The fusion proteinsare secreted into the culture supernatant, which is harvested for use incytokine assays, such as interferon-γ (IFN-γ) secretion assays (Martin,et al., J. Immunol. 167:3668-3676 (2001)).

PBMCs are activated with a suboptimal concentration of soluble CD3 andvarious concentrations of purified, soluble anti-peroxidasin-likeprotein monoclonal antibody or control IgG. For peroxidasin-likeprotein-Ig cytokine assays, anti-human Fc Ig at 5 or 20 μg/ml is boundto 96-well plates and incubated overnight at 4° C. Excess antibody isremoved and either peroxidasin-like protein-Ig or control Ig is added at20-50 μg/ml and incubated for 4 h at room temperature. The plate iswashed to remove excess fusion protein before adding cells and anti-CD3to various concentrations. Supernatants are collected after 48 h ofculture and IFN-γ levels are measured by sandwich ELISA, using primaryand biotinylated secondary anti-human IFN-γ antibodies as recommended bythe manufacturer.

Example 54 Diagnostic Methods Using Peroxidasin-Like Protein-SpecificAntibodies to Detect Peroxidasin-Like Protein Expression

Expression of peroxidasin-like protein in tissue samples (normal ordiseased) is detected using anti-peroxidasin-like protein antibodies.Samples are prepared for immunohistochemical (IHC) analysis by fixingthe tissue in 10% formalin embedding in paraffin, and sectioning usingstandard techniques. Sections are stained using the peroxidasin-likeprotein-specific antibody followed by incubation with a secondary horseradish peroxidase (HRP)-conjugated antibody and visualized by theproduct of the HRP enzymatic reaction.

Expression of peroxidasin-like protein on the surface of cells within ablood sample is detected by flow cytometry. Peripheral blood mononuclearcells (PBMC) are isolated from a blood sample using standard techniques.The cells are washed with ice-cold PBS and incubated on ice with theperoxidasin-like protein-specific polyclonal antibody for 30 min. Thecells are gently pelleted, washed with PBS, and incubated with afluorescent anti-rabbit antibody for 30 min. on ice. After theincubation, the cells are gently pelleted, washed with ice cold PBS, andresuspended in PBS containing 0.1% sodium azide and stored on ice untilanalysis. Samples are analyzed using a FACScalibur flow cytometer(Becton Dickinson) and CELLQuest software (Becton Dickinson). Instrumentsetting are determined using FACS-Brite calibration beads(Becton-Dickinson).

Tumors expressing peroxidasin-like protein are imaged usingperoxidasin-like protein-specific antibodies conjugated to aradionuclide, such as ¹²³I, and injected into the patient for targetingto the tumor followed by X-ray or magnetic resonance imaging.

Example 55 Tumor Imaging Using Peroxidasin-Like Protein-SpecificAntibodies

Peroxidasin-like protein-specific antibodies are used for imagingperoxidasin-like protein-expressing cells in vivo. Six-week-old athymicnude mice are irradiated with 400 rads from a cesium source. Three dayslater the irradiated mice are inoculated with 4×10⁷ RA1 cells and 4×10⁶human fetal lung fibroblast feeder cells subcutaneously in the thigh.When the tumors reach approximately 1 cm in diameter, the mice areinjected intravenously with an inoculum containing 100 μCi/10 μg of¹³¹I-labeled peroxidasin-like protein-specific antibody. At 1, 3, and 5days postinjection, the mice are anesthetized with a subcutaneousinjection of 0.8 mg sodium pentobarbital. The immobilized mice are thenimaged in a prone position with a Spectrum 91 camera equipped with apinhole collimator (Raytheon Medical Systems; Melrose Park, Ill.) set torecord 5,000 to 10,000 counts using the Nuclear MAX Plus image analysissoftware package (MEDX Inc.; Wood Dale, Ill.) (Hornick, et al., Blood89:4437-4447 (1997)).

1. An isolated polynucleotide comprising a nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 1-4, 6, 14, 16, 25-27, 29,157-159, 161, 183-185, 187, 214, 216, 240, 242, 271, 273, 300-301, 303,322, 324, 345-347, 349, 353-354, 356, 377, 379, 405-407, 409,418-419,421, 441443, 485-486, 488, 503, 504, 506, 514-515, 517, 526-527,529, 547, 549, 556, 558, 570-571,573,577-578,580,587,589, 601, 603, 606,608, 611, 613, 617, 619, 621, 623, 625, 627, 629, or 631 or the matureprotein coding portion thereof.
 2. An isolated polynucleotide encoding apolypeptide with biological activity, wherein said polynucleotidehybridizes to the polynucleotide of claim 1 under stringenthybridization conditions (0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS),1 mM EDTA at 65° C.).
 3. The polynucleotide of claim 1 wherein saidpolynucleotide is DNA.
 4. An isolated polynucleotide which comprises thecomplement of any one of the polynucleotides of claim
 1. 5. A vectorcomprising the polynucleotide of claim
 1. 6. An expression vectorcomprising the polynucleotide of claim
 1. 7. A host cell geneticallyengineered to comprise the polynucleotide of claim
 1. 8. A host cellgenetically engineered to comprise the polynucleotide of claim 1operatively associated with a regulatory sequence that modulatesexpression of the polynucleotide in the host cells.
 9. An isolatedpolypeptide, wherein the polypeptide is selected from the groupconsisting of: (a) a polypeptide encoded by any one of thepolynucleotides of claim 1; and (b) a polypeptide encoded by apolynucleotide hybridizing under stringent conditions with any one ofSEQ ID NO: 5, 7-13, 15, 17-24, 28, 30-156, 160, 162-182, 186, 188-213,215, 217-239, 241, 243-270, 272, 274-299, 302, 304-321, 323, 325-344,348, 350-352, 355, 357-376, 378, 380-401, 408, 410-414, 415, 420,422-439, 444-480, 482-484, 487, 489-501, 505, 507-512, 516, 518-524,528, 530-539, 542, 544-546, 548, 550-553, 557, 559-567, 572, 574, 576,579, 581-584, 588, 590, 596, 602, 604-605, 607, 609-610, 612, 614-615,618, 620, 622, 624, 626, 628, 630, 632, or 634-653.
 10. An isolatedpolypeptide comprising an amino acid sequence selected from the groupconsisting of any one of the polypeptides of SEQ ID NO: 5, 7-13, 15,17-24, 28, 30-156, 160, 162-182, 186, 188-213, 215, 217-239, 241,243-270, 272, 274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355,357-376, 378, 380-401, 408, 410-414, 415, 420, 422-439, 444-480,482-484, 487, 489-501, 505, 507-512, 516, 518-524, 528, 530-539, 542,544-546, 548, 550-553, 557, 559-567, 572, 574, 576, 579, 581-584, 588,590, 596, 602, 604-605, 607, 609-610, 612, 614-615, 618, 620, 622, 624,626, 628, 630, 632, or 634-653.
 11. A composition comprising thepolypeptide of claim 9 or 10 and a carrier.
 12. An antibody directedagainst the polypeptide of claim 9 or
 10. 13. A method for detecting thepolynucleotide of claim 1 in a sample, comprising the steps of: (a)contacting the sample with polynucleotide probe that specificallyhybridizes to the polynucleotide under conditions which permit formationof a probe/polynucleotide complex; and (b) detecting the presence of aprobe/polynucleotide complex, wherein the presence of the complexindicates the presence of a polynucleotide.
 14. A method for detectingthe polynucleotide of claim 1 in a sample, comprising the steps of: (a)contacting the sample under stringent hybridization conditions withnucleic acid primers that anneal to the polynucleotide of claim 1 undersuch conditions; and (b) amplifying the polynucleotide or fragmentthereof, so that if the polynucleotide or fragment is amplified, thepolynucleotide is detected.
 15. The method of claim 14, wherein thepolynucleotide is an RNA molecule that encodes the polypeptide of claim9 or 10, and the method further comprises reverse transcribing anannealed RNA molecule into a cDNA polynucleotide.
 16. A method ofdetecting the presence of the polypeptide of claim 9 or 10 having theamino acid sequence of any one of SEQ ID NO: 5, 7-13, 15, 17-24, 28,30-156, 160, 162-182, 186, 188-213, 215, 217-239, 241, 243-270, 272,274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355, 357-376, 378,380-401, 408, 410-414, 415, 420, 422-439, 444-480, 482-484, 487,489-501, 505, 507-512, 516, 518-524, 528, 530-539, 542, 544-546, 548,550-553, 557, 559-567, 572, 574, 576, 579, 581-584, 588, 590, 596, 602,604-605, 607, 609-610, 612, 614-615, 618, 620, 622, 624, 626, 628, 630,632, or 634-653 or a fragment thereof in a cell, tissue or fluid samplecomprising: (a) contacting said cell, tissue or fluid sample with anantibody or fragment of claim 10 under conditions which permit theformation of an antibody/polypeptide complex; and (b) detecting thepresence of an antibody/polypeptide complex, wherein the presence of theantibody/polypeptide complex indicates the presence of any of thepolypeptides of claim
 10. 17. A method for identifying a compound thatbinds to a polypeptide of any one of SEQ ID NO: 5, 7-13, 15, 17-24, 28,30-156, 160, 162-182, 186, 188-213, 215, 217-239, 241, 243-270, 272,274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355, 357-376, 378,380-401, 408, 410-414, 415, 420, 422439, 444480, 482-484, 487, 489-501,505, 507-512, 516, 518-524, 528, 530-539, 542, 544-546, 548, 550-553,557, 559-567, 572, 574, 576, 579, 581-584,588,590,596, 602,604-605,607,609-610,612,614-615,618,620,622, 624, 626, 628, 630, 632, or634-653 comprising: (a) contacting a compound with the polypeptide ofany of SEQ ID NO: 5, 7-13, 15, 17-24, 28, 30-156, 160, 162-182, 186,188-213, 215, 217-239, 241, 243-270, 272, 274-299, 302, 304-321, 323,325-344, 348, 350-352, 355, 357-376, 378, 380-401, 408, 410-414, 415,420, 422-439, 444-480, 482-484, 487, 489-501, 505, 507-512, 516,518-524, 528, 530-539, 542, 544-546, 548, 550-553, 557, 559-567, 572,574, 576, 579, 581-584, 588, 590, 596, 602, 604-605, 607, 609-610, 612,614-615, 618, 620, 622, 624, 626, 628, 630, 632, or 634-653 for a timesufficient to form a polynucleotide/compound complex; and (b) detectingthe complex, so that if a polypeptide/compound complex is detected, acompound that binds to any one of SEQ ID NO: 5, 7-13, 15, 17-24, 28,30-156, 160, 162-182, 186, 188-213, 215, 217-239, 241, 243-270, 272,274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355, 357-376, 378,380-401, 408, 410-414, 415, 420, 422-439, 444-480, 482-484, 487,489-501, 505, 507-512, 516, 518-524, 528, 530-539, 542, 544-546, 548,550-553, 557, 559-567, 572, 574, 576, 579, 581-584, 588, 590, 596, 602,604-605, 607, 609 610, 612, 614-615, 618, 620, 622, 624, 626, 628, 630,632, or 634-653 is identified.
 18. A method for identifying a compoundthat binds to any one of the polypeptides of SEQ ID NO: 5, 7-13, 15,17-24, 28, 30-156, 160, 162-182, 186, 188-213, 215, 217-239, 241,243-270, 272, 274-299, 302, 304-321, 323, 325-344, 348, 350-352, 355,357-376, 378, 380-401, 408, 410-414, 415, 420, 422-439, 444-480,482-484, 487, 489-501, 505, 507-512, 516, 518-524, 528,530-539,542,544-546,548,550-553, 557, 559-567, 572, 574, 576, 579,581-584, 588, 590, 596, 602, 604-605, 607, 609-610, 612, 614-615, 618,620, 622, 624, 626, 628, 630, 632, or 634-653, comprising: (a)contacting a compound with the polypeptide of any one of SEQ ID NO: 5,7-13, 15, 17-24, 28, 30-156, 160, 162-182, 186, 188-213, 215, 217-239,241, 243-270, 272, 274-299, 302, 304-321, 323, 325-344, 348, 350-352,355, 357-376, 378, 380-401, 408, 410-414, 415, 420, 422-439, 444-480,482-484, 487, 489-501, 505, 507-512, 516, 518-524, 528, 530-539, 542,544-546, 548, 550-553, 557, 559-567, 572, 574, 576, 579, 581-584, 588,590, 596, 602, 604-605, 607, 609-610, 612, 614-615, 618, 620, 622, 624,626, 628, 630, 632, or 634-653, in a cell, for a time sufficient to forma polypeptide/compound complex, wherein the complex drives theexpression of a reporter gene sequence in the cell; and (b) detectingthe complex by detecting reporter gene sequence expression, so that if apolypeptide/compound complex is detected, a compound that binds to anyone of the polypeptides of SEQ ID NO: 5, 7-13, 15, 17-24, 28, 30-156,160, 162-182, 186, 188-213, 215, 217-239, 241, 243-270, 272, 274-299,302, 304-321, 323, 325-344, 348, 350-352, 355, 357-376, 378, 380401,408, 410-414, 415, 420, 422-439, 444-480, 482-484, 487, 489-501, 505,507-512, 516, 518-524, 528, 530-539, 542, 544-546, 548, 550-553, 557,559-567, 572, 574, 576, 579, 581-584, 588, 590, 596, 602, 604-605, 607,609-610, 612, 614-615, 618, 620, 622, 624, 626, 628, 630, 632, or634-653 is identified.
 19. A method of producing the polypeptides ofclaim 9 or 10, comprising: (a) culturing the host cell of claim 7 or 8for a period of time sufficient to express the polypeptide; and (b)isolating the polypeptide from the cell or culture media in which thecell is grown.
 20. A kit comprising any one of the polypeptides of claim9 or
 10. 21. A nucleic acid array comprising the polynucleotide of claim1 attached to a surface.
 22. The polypeptide of claim 9 or 10 whereinthe polypeptide is provided on a polypeptide array.
 23. A method formodifying the proliferation of neural cells, comprising the step ofadministering a composition to said cells in an amount effective tomodify the proliferation of said cells, wherein said composition is anNgRHy polypeptide.
 24. The method of claim 21, wherein said modifying isinducing the proliferation of neural cells.
 25. The method of claim 21,wherein said modifying is inhibiting the proliferation of neural cells.